Philipp S. Muether

Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruchs membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3.

Cumulative Effect of Risk Alleles in CFH, ARMS2, and VEGFA on the Response to Ranibizumab Treatment in Age-Related Macular Degeneration

PURPOSE Intravitreal ranibizumab injections currently are the standard treatment for neovascular age-related macular degeneration (AMD). However, a broad range of response rates have been observed, the reasons for which are poorly understood. This pharmacogenetic study evaluated the impact of high-risk alleles in CFH, ARMS2, VEGFA, vascular endothelial growth factor (VEGF) receptor KDR, and genes involved in angiogenesis (LRP5, FZD4) on the response to ranibizumab treatment and on the age of treatment onset. In contrast to previous studies, the data were stratified according to the number of high-risk alleles to enable the study of the combined effects of these genotypes on the treatment response. DESIGN Case series study. PARTICIPANTS A cohort of 420 eyes of 397 neovascular AMD patients. METHODS The change in visual acuity (VA) between baseline and after 3 ranibizumab injections was calculated. Genotyping of single nucleotide polymorphisms in the CFH, ARMS2, VEGFA, KDR, LPR5, and FZD4 genes was performed. Associations were assessed using linear mixed models. MAIN OUTCOME MEASURES The VA change after 3 ranibizumab injections and the age of neovascular disease onset. RESULTS After ranibizumab treatment, AMD patients without risk alleles in the CFH and ARMS2 genes (4.8%) demonstrated a mean VA improvement of 10 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, whereas no VA improvement was observed in AMD patients with 4 CFH and ARMS2 risk alleles (6.9%; P = 0.014). Patients with 4 high-risk alleles in CFH and ARMS2 were 5.2 years younger than patients with 1 or 2 risk alleles, respectively (63.5%; P<0.0001). The mean age at which the first ranibizumab treatment was carried out among AMD patients with all 6 risk alleles in CFH, ARMS2, and VEGFA was 65.9 years (2%) versus 75.3 years in patients with 0 or 1 high-risk allele (8.8%; P = 0.001). After ranibizumab treatment, patients with 6 high-risk alleles demonstrated a mean VA loss of 10 ETDRS letters (P<0.0001). CONCLUSIONS This study evaluated the largest pharmacogenetic AMD cohort reported to date. A cumulative effect of high-risk alleles in CFH, ARMS2, and VEGFA seems to be associated with a younger age of onset in combination with poor response rates to ranibizumab treatment.

Serum concentrations of vascular endothelial growth factor in an infant treated with ranibizumab for retinopathy of prematurity.

ganglion cells were first discovered 10 years ago, and many important aspects of their function remains unknown, not least the important question of how much or how little light is needed to maintain stable photoentrainment. Melanopsin-driven photoentrainment of circadian rhythms and pupillary response to short wavelength light are coupled, and looking at the pupillary light response to blue light is a clinically useful indirect measurement of the function of the melanopsin containing retinal ganglion cells. Melanopsin is a bi-stable molecule (Mure et al. 2009) that is reflected in the enhanced pupillary constriction to blue light after prior exposure to blue light (Hansen et al. 2011). Pupillary blue light response depends on pupil size and is greater after pupillary dilation (Nissen et al. 2011). Yet surprisingly, melanopsinmediated pupillary blue light response is enhanced with age in spite of age-related miosis and reduced lens transmission (Herbst et al. 2012). Speculatively, the system may adapt to decreased blue light levels with age by increasing the sensitivity. These observations demonstrate the complexity of the system and difficulties encountered when extrapolating from the filtering effects of the lens or IOLs to the photoentrainment potential of the aged or even pseudophakic eye. We felt there were too many unknown factors to compute a photoentrainment potential for the aged eye and hence restricted our computations to the effect of the lens. One aspect of the debate is whether blue-blocking IOLs are detrimental to photoentrainment of circadian rhythms or not. Equally important is the question of whether to use blue-blocking IOLs or not. The main rationale for using blueblocking IOLs is the theory that blue light is involved in the pathogenesis of age-related macular degeneration (AMD). As we write in our paper, the theory has not been scientifically proven and concluding that we argue that the ‘chromophores they consider to be optically insignificant to adversely affect circadian photoentrainment might somehow reduce the risk of AMD’ is a twisting of our argumentation. What we do write is that if someone were to conduct a study proving a protective effect of blue-blocking IOLs on AMD, the study would need to have a considerable time perspective. We do not yet have the scientific proof to say whether blue-blocking IOLS do or do not reduce the risk of AMD. Sleep disturbances increase with age, and a correlation with lens yellowing has been found (Kessel et al. 2011). Cataract surgery (with UV-blocking IOLs) leads to better sleep and well-being (Asplund & Lindblad 2004) indicating that short wavelength filtering by the lens is important for the photoentrainment of circadian rhythms in vivo. Our results showed that the difference of yellow-tinted and UV-only blocking IOLs is exceeded by several folds by the natural discoloration of the human lens. How this may affect photoentrainment is – of course – speculation and as Dr. Mainster and Dr. Turner states prospective, randomized clinical trials are needed to determine the clinical significance.

Vascular Endothelial Growth Factor in Patients with Exudative Age-related Macular Degeneration Treated with Ranibizumab

OBJECTIVES To analyze the temporal correlations of vascular endothelial growth factor (VEGF) suppression, morphologic recurrence of choroidal neovascularization (CNV), and visual acuity loss in eyes with exudative age-related macular degeneration (AMD) treated with ranibizumab. DESIGN Nonrandomized, prospective, clinical study. PARTICIPANTS Forty-seven eyes of 47 patients with exudative AMD undergoing intravitreal ranibizumab injections. METHODS Aqueous humor specimens were taken before each intravitreal ranibizumab injection. Visual acuity testing, spectral domain optical coherence tomography (SD-OCT), and fundoscopy were performed before each injection. Vascular endothelial growth factor A was measured by Luminex multiplex bead analysis (Luminex Inc., Austin, TX). MAIN OUTCOME MEASURES Intraocular VEGF concentration, recurrence of CNV activity shown by SD-OCT, and vision loss. RESULTS Ranibizumab resulted in complete VEGF suppression within a mean period of 37.8 days (standard deviation [SD] ± 4.8 days; range, 26-49 days). Recurrences of CNV activity as determined by SD-OCT occurred 93.7 days (SD ± 69.9 days; range, 57-368 days) after the last ranibizumab treatment. The VEGF levels were never suppressed when a recurrence occurred. Functional recurrence (visual acuity) occurred 114.3 days (SD ± 81.4 days; range, 57-398 days) after previous treatment. The VEGF levels did not differ significantly between baseline and recurrence (69.3 pg/ml vs. 74.14 pg/ml; 95% confidence interval, -18.87 to 9.12). CONCLUSIONS A monthly intravitreal injection of 0.5 mg ranibizumab yields a durable VEGF inhibition. The recurrences of CNV as determined by SD-OCT are always preceded by a loss of intraocular VEGF suppression and usually followed by loss of visual acuity in the further course.

Suppression of Intraocular Vascular Endothelial Growth Factor During Aflibercept Treatment of Age-Related Macular Degeneration

PURPOSE To determine the duration of suppression of aqueous humor concentrations of vascular endothelial growth factor (VEGF) in eyes with neovascular age-related macular degeneration (AMD) treated with aflibercept. DESIGN Nonrandomized prospective clinical study. METHODS Twenty-seven eyes of 27 neovascular AMD patients receiving intravitreal aflibercept injections on a pro re nata regimen driven by spectral-domain optical coherence tomography (SD OCT) were included in this study. A total of 132 aqueous humor specimens were collected before intravitreal aflibercept injections and their VEGF-A concentrations assayed by multiplex bead analysis. RESULTS Mean aqueous humor VEGF concentrations before treatment initiation were 90.6 ± 37.1 pg/mL (range 23.4-190.3 pg/mL). Intravitreal injection of aflibercept suppressed the aqueous VEGF concentrations to below the lower limit of quantification (<4 pg/mL) in all patients. The mean duration of VEGF suppression below the lower limit of quantification was >71 ± 18 days. The earliest time after injection at which the VEGF concentration recovered to above the lower limit of quantification was 55 days in 1 patient and >56 days, the recommended aflibercept treatment interval, in 20 patients. The aqueous VEGF recovery status of 6 patients was uncertain after 56 days. CONCLUSIONS On average, VEGF concentrations in the aqueous humor were suppressed below the lower limit of quantification after intravitreal aflibercept injections for about 10 weeks. This aqueous suppression time suggests durable VEGF inhibition for most patients dosed with aflibercept every 8 weeks.

Modulation of Hypoxia-Induced Neovascularization by JSM6427, an Integrin α5β 1 Inhibiting Molecule

Purpose: Integrin α 5β 1, a fibronectin receptor, is involved in endothelial cell migration and proliferation. Here we investigate the effect of JSM6427, an integrin α 5β 1 inhibiting molecule, on the development of retinal vascular system using the mouse model of oxygen-induced retinopathy (OIR). Methods: Endothelial cell migration and sprouting was analyzed in vitro using a 2D migration assay and a 3D sprouting/angiogenesis assay in fibrin gel. C57BL/C6 mice were exposed to 75% oxygen from postnatal day 7 (P7) to P12 and returned to room air thereafter. Intravitreal injection of 40 μ g JSM6427 was performed in each one eye on P14. On P17, vascular area, avascularized area, and neovascular blood vessel tufts were quantified after perfusion with fluorescein-coupled concanavalin A. The number of retinal neovascular cell nuclei was determined in hematoxylin-stained cross sections of the eyes. Integrin α 5 expression was determined by immunohistochemistry. Results: In vitro, JSM6427 inhibits the migration of HUVEC and the tube formation induced by both bFGF and VEGF. In vivo, integrin α 5 expression was detectable in neovascular retinal blood vessels. Oxygen treatment (positive control) in comparison with no oxygen treatment (negative control) reduced significantly the vascularized area and increased the avascularized area. A single intravitreal injection of 40 μ g JSM6427 resulted in a significant reduction of the vascularized area and the number of preretinal nuclei in comparison with the intravitreal injection of the vehicle while the avascularized area increased significantly. Conclusions: These results imply an essential role of integrin α 5β 1 in the refining of the retinal vasculature in OIR and suggest JSM6427 may have a possible therapeutic function for neovascular disease.

Intraocular growth factors and cytokines in patients with dry and neovascular age-related macular degeneration.

Purpose: To analyze intraocular growth factor and cytokine concentrations in eyes with different stages of age-related macular degeneration (AMD) compared with controls. Methods: The Clinical Age-Related Maculopathy Staging (CARMS) system was used for assignment of patients into the respective categories. Aqueous humor specimens were taken before cataract surgery in 21 controls (CARMS 1) and in 17 early (CARMS 2) and 16 intermediate (CARMS 3) AMD patients. In 18 neovascular (CARMS 5) AMD patients, specimens were taken immediately before anti–vascular endothelial growth factor intravitreal therapy. Luminex multiplex bead assays were conducted for endostatin, angiogenin, vascular endothelial growth factor, platelet-derived growth factor AA, placental growth factor, thrombospondin 2, and fibroblast growth factor a. Results: Vascular endothelial growth factor concentrations were elevated in CARMS 3 (P = 0.037) and tended to be elevated in CARMS 5 (P = 0.093), whereas levels in CARMS 2 (P = 0.425) were similar to CARMS 1. Platelet-derived growth factor levels were diminished in CARMS 2 (P = 0.020), with a trend to lower levels for CARMS 3 (P = 0.099) and CARMS 5 (P = 0.082) compared with CARMS 1. For CARMS 5, antiangiogenic endostatin was elevated (P < 0.002), while antiangiogenic thrombospondin 2 was reduced (P = 0.029). Conclusion: Clinical Age-Related Maculopathy Staging 3 dry AMD was associated with higher vascular endothelial growth factor levels than CARMS 5 neovascular AMD. Therefore, intraocular vascular endothelial growth factor concentrations do not seem to reflect choroidal neovascularization activity in neovascular AMD directly. Platelet-derived growth factor was decreased in most forms of AMD. The antiangiogenic endostatin was exclusively elevated in neovascular AMD, while thrombospondin 2 was reduced. Age-related macular degeneration disease seems to be associated with a generally altered cytokine system.

Long-term stability of vascular endothelial growth factor suppression time under ranibizumab treatment in age-related macular degeneration.

PURPOSE To determine intra-individual long-term stability of vascular endothelial growth factor (VEGF) suppression time in eyes with neovascular age-related macular degeneration (AMD) treated with ranibizumab. DESIGN Nonrandomized, prospective clinical study. METHODS Eighty-three eyes of 83 patients with neovascular AMD undergoing intravitreal ranibizumab injections were included in the study. A total of 859 aqueous humor specimens were taken before each intravitreal ranibizumab injection. Vascular endothelial growth factor A was measured by multiplex bead analysis. RESULTS Ranibizumab resulted in complete VEGF suppression within a mean period of 36.4 days (standard deviation ±6.7 days; range, 26-69 days). Intra-individual suppression time was stable within a period of up to 3 years. Among 859 VEGF measurements, only 5 (0.58%) deviated from this pattern. Nonsuppressed VEGF levels did not differ significantly between baseline and recurrence (68.0 pg/mL vs 69.3 pg/mL) and did not correlate with choroidal neovascularization size and lesion type. CONCLUSIONS Both the long-term stability and the broad range of individual suppression times after ranibizumab injections would allow and justify adjustment of continuous injections individually in order to achieve permanent VEGF suppression in patients.

Upregulation of TGF-ß1 in experimental proliferative vitreoretinopathy is accompanied by epithelial to mesenchymal transition

BackgroundProliferative vitreoretinopathy (PVR) is characterized by epithelial to mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells and consecutive formation of fibrous membranes, leading to retinal redetachment. Transforming growth factor beta (TGF-ß) has been suggested to play an important role in this process, but the role of TGF-ß isoforms is unknown.MethodsIn pigmented rabbits (n = 14), PVR was induced by cryopexy and a full-thickness limbus-parallel incision. PVR was evaluated by indirect ophthalmoscopy. Concentrations of TGF-ß isoforms were determined by multiplex bead assay analysis in aqueous humor (AH) and vitreous samples. EMT marker vimentin was analyzed by western blot. Masson’s-trichrome, haematoxilin and eosine (H&E), and immunohistochemical analysis for EMT marker alpha SMA were performed on cross-sections of eyes.ResultsPVR was induced in all treated eyes. The number of quadrants affected by PVR was 1 (n = 5), 2 (n = 2), 3 (n = 2), 4 (n = 5). Vimentin and alpha SMA were expressed during PVR development. During PVR development, both TGF-ß1 levels (AH: p = 0.001; vitreous: p = 0.002) and TGF-ß2 levels increased (AH: p = 0.027; vitreous: p = 0.02), while TGF-ß3 was not detected at any timepoint. The increase was more pronounced for TGF-ß1 than for TGF- ß2 (AH: p = 0.002; vitreous: p = 0.0005), and only TGF-ß1 correlated with the amount of PVR (p = 0.024, r = 0,723).ConclusionsDevelopment of PVR membranes was accompanied by a pronounced upregulation of TGF-ß1, rather than TGF-ß2. Therefore TGF-ß1 could be a promising target for inhibition of PVR.

Profibrotic cytokines in aqueous humour correlate with aqueous flare in patients with rhegmatogenous retinal detachment.

Background Aqueous flare as determined by laser flare photometry in the anterior chamber is a strong preoperative predictor for proliferative vitreoretinopathy (PVR) in patients with primary retinal detachment (RD). We analysed various cytokines in aqueous humour samples in relation to aqueous flare and postoperative PVR incidence in patients with RD. Methods Preoperatively, the aqueous flare of patients with RD was measured quantitatively with a laser flare metre and aqueous humour samples were collected and analysed for interferon γ, tumour necrosis factor α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, vascular endothelial growth factor (VEGF)-A, platelet derived growth factor (PDGF)-aa, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, fibroblast growth factor (FGF)-aa and FGF-bb by multiplex fluorescent bead-based immunoassays. Three months after RD surgery patients were examined for PVR development. Results Of 67 consecutive patients, 10 developed at least PVR grade C. Patients with flare values >15 pc/ms (n=20) and the 10 patients with postoperative PVR all had significantly elevated levels of IL-6, IL-8, MCP-1 and TGF-β1 in aqueous humour (p≤0.05). Levels of VEGF-A, PDGF-aa and TGF-β2 were not significantly changed. Other cytokines were below the detection threshold. Eight of the 10 patients (80%) with PVR had elevated flare values of >15 pc/ms and 8 of the 20 patients (40%) with flare >15 pc/ms developed PVR. The OR for PVR with flare values >15 pc/ms was 30.7 (p=0.0001). Conclusions Laser flare photometry allows simple risk estimation for later PVR development. Elevated laser flare values correspond to an altered profibrotic intraocular cytokine milieu. These factors therefore constitute promising targets for a prophylactic intervention.