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 Bruch's 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

Clinical correlation to differences in ranibizumab and aflibercept vascular endothelial growth factor suppression times

Aim To determine clinical correlations to intraocular vascular endothelial growth factor A (VEGF-A) suppression times (VSTs) on the treatment of neovascular age-related macular degeneration (nAMD) with ranibizumab (Lucentis) or aflibercept (Eylea). Methods Seven of 89 treatment-naive nAMD eyes showed persistent choroidal neovascular membrane (CNV) activity throughout a spectral domain optical coherence tomography (SD-OCT)-driven pro re nata (PRN) regimen of intravitreal ranibizumab injections over 284months. The treatment was switched to PRN aflibercept injections and patients were followed for another 152months. A total of 160 aqueous humour specimens were collected before the intravitreal injections, and their VEGF-A concentrations were assayed by Luminex multiplex bead analysis (Luminex, Austin, Texas, USA). Intraocular VEGF-A concentrations were correlated to CNV activity shown by SD-OCT. Results The mean duration of suppression of VEGF-A concentrations in aqueous humour below the lower limit of quantification of our assay was 34 +/- 5 (26-69) days for ranibizumab and 67 +/- 14 (49-89) days for aflibercept (p<0.001). The percentual reduction of central retinal volume (CRV) 6weeks after injection was higher for aflibercept compared with ranibizumab (p=0.009). The time point of clinical re-activity occurred about 50% earlier than the respective VST for each ranibizumab and aflibercept. Conclusions The VST under aflibercept treatment exceeded that under ranibizumab treatment by a factor of 2. This difference correlated with differential clinical CRV reduction 6weeks after the respective injection. For both medications, clinical activity was found at a time point as early as 50% of the individual VST. Trial registration number NCT01213667, post-result

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 (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. (C) 2014 by Elsevier Inc. All rights reserved

A model of the ocular pharmacokinetics involved in the therapy of neovascular age-related macular degeneration with ranibizumab

Background To develop a model of the pharmacokinetics of vascular endothelial growth factor (VEGF-A) determined in samples of aqueous humour from patients with neovascular age-related macular degeneration (AMD) treated with ranibizumab (Lucentis). Methods Post hoc analysis of data from 31 eyes of 31 patients with AMD treated with ranibizumab gathered in a non-randomised, prospective clinical study. VEGF-A concentrations were measured in 440 aqueous humour samples by Luminex multiplex bead analysis (Luminex, Austin, Texas, USA). Results The kinetics of recovery of VEGF-A from suppression by ranibizumab were well described by a simple model: VEGF-A is produced at a constant individual rate; VEGF-A and ranibizumab disperse rapidly within the vitreous chamber and bind with a known affinity; both are eliminated at identical rates from the vitreous chamber in a constant but individual flow into the anterior chamber, and are finally cleared by draining into the peripheral circulation. Average rates of VEGF-A production were predicted to be 5.8 fmol/day (range: 2.7-10.1 fmol), and elimination half-times predicted to be 3.5 days (range: 2.3-5.5 days). The duration of complete VEGF-A suppression in the aqueous humour averaged 41 days (range: 28-67 days). Conclusions The ocular pharmacokinetics of VEGF-A and ranibizumab have been linked for the first time in a simple and plausible model which suggests that it might be possible to anticipate individual VEGF-A suppression times

Vascular endothelial growth factor suppression times in patients with diabetic macular oedema treated with ranibizumab

Background To measure vascular endothelial growth factor (VEGF) levels in aqueous humour from patients with diabetic macular oedema (DME) treated with ranibizumab and to determine how long VEGF was suppressed. Methods In this nonrandomised, prospective clinical study, 17 eyes of 17 patients were included in the study. A total of 110 aqueous humour samples were taken before an intravitreal ranibizumab injection in patients with DME. VEGF-A was measured by Luminex multiplex bead analysis (Luminex Inc, USA). Results VEGF was completely suppressed in all patients after ranibizumab injections for a mean of 33.7 days (SD +/- 5.1, range 27-42, median 34). VEGF suppression times were individually stable during the observation time of up to 16 months. There was no statistically significant difference of VEGF levels at baseline and before the beginning of a new injection series (123.6 pg/mL vs 125.1 pg/mL; p=1.0, Wilcoxon). Conclusions Monthly ranibizumab injections lead to a complete VEGF suppression in patients with DME. The long-term stability and the range of suppression times among individuals suggest that some patients could benefit from individual injection intervals

COURSE OF INTRAOCULAR PRESSURE AFTER VITREORETINAL SURGERY Is Early Postoperative Intraocular Pressure Elevation Predictable?

Purpose: Assessment of intraocular pressure (IOP) after vitreoretinal surgery is important to ensure functionality of the eye. Incidences and risk factors for early postoperative IOP elevation were evaluated. Methods: In a prospective case series of 210 vitreoretinal cases, IOP-lowering treatment was performed at IOP values of >= 30 mmHg. Differences in IOP elevation in relation to surgical procedures and tamponades were evaluated. Results: Sixty-two patients required treatment (29.5%). Encircling bands were associated with a high risk for IOP elevation when combined with pars plana vitrectomy and gas tamponade (37.9%) or oil tamponade (50.0%). Panretinal laser photocoagulation in conjunction with oil tamponade for proliferative diabetic retinopathy traction retinal detachment (RD) resulted in the highest risk for IOP increases (83.3%). Intraocular pressure elevation in proliferative diabetic retinopathy traction RD often evolved 4 hours after vitreoretinal surgery compared with 8 hours to 12 hours after vitreoretinal surgery in rhegmatogenous RD. Silicone oil removals (7.1%) and external buckling procedures (0.0%) carried low risks for IOP increases. Conclusion: Patients treated for proliferative diabetic retinopathy traction RD and for primary rhegmatogenous RD were at high risk for prolonged IOP elevation. These groups also required medical retreatment most often and should therefore be closely monitored. Care must be taken not to overlook delayed IOP elevations. RETINA 31:1545-1552, 201

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

To 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. This 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. Eyes 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. POAG 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

A Randomized Controlled Clinical Trial Comparing 20 Gauge and 23 Gauge Vitrectomy for Patients with Macular Hole or Macular Pucker

IntroductionTo compare the transconjunctival sutureless 23 gauge (G) pars plana vitrectomy (PPV) with 20 G PPV regarding inflammation, safety, visual outcome and patient comfort.MethodsWe included 103 patients with symptomatic macular hole or macular pucker, scheduled for vitrectomy in this prospective, randomized, controlled, mono-center clinical trial. Patients were randomized 1:1 to either 20G PPV (n=51) or 23G PPV (n=52). All eyes underwent standard 20G or 23G PPV with membrane peeling. Primary outcome measure was change in aqueous humor flare 3weeks after surgery compared with baseline. Secondary outcome measures were flare values 2days and 26weeks after surgery, subjective discomforts measured with a visual analog scale, best-corrected visual acuity, duration of surgery, intraocular pressure (IOP) and adverse events.ResultsThere was no significant difference in change of flare 3weeks after PPV [- 1.7, 95% CI (- 6.3 to 2.9), p=0.466]. Both groups showed a significant increase in flare 2 days after surgery (20G: p<0.001, 23G: p=0.002), but only the 20G group after 3weeks (p=0.011). The gain in visual acuity after 3 weeks was higher after 23G PPV (4.2 95% CI (0.4-8.0, p=0.029), but without a difference after 6months. The duration of surgery was shorter in the 23G group (p<0.001). Patient comfort 3weeks after surgery was greater after 23G PPV (foreign body sensation p=0.002; itching: p=0.021). However, the rate of complications did not differ between the groups.ConclusionThe primary aim, showing the superiority of the 23G group regarding the change of flare value from baseline to 3weeks after surgery, was not met, but the level of inflammation decreased faster after 23G PPV. Clear advantages of the 23G PPV were a lower risk of postoperative IOP elevation, a shorter surgery time, faster visual recovery and greater patient comfort in the early postoperative phase.Clinical Trial Registration NumberClinicalTrials.gov NCT01969929

LONG-TERM ALTERATIONS OF SYSTEMIC VASCULAR ENDOTHELIAL GROWTH FACTOR LEVELS IN PATIENTS TREATED WITH RANIBIZUMAB FOR AGE-RELATED MACULAR DEGENERATION

Purpose: To analyze long-term changes of systemic vascular endothelial growth factor (VEGF) levels in patients treated with ranibizumab for neovascular age-related macular degeneration. Methods: Sixty-one patients with neovascular age-related macular degeneration and 68 age-matched controls were included in the study. Patients were treated with ranibizumab on a pro re nata regimen. Plasma samples were collected before initiation of treatment and after 1 year (30 patients) or 2 years (31 patients) of treatment. Vascular endothelial growth factor was measured by Luminex microbead analysis. Results: At baseline, patients with neovascular age-related macular degeneration and controls did not differ significantly in VEGF levels (P = 0.062). There was a significant decline in systemic VEGF levels of 39.5% after 1 year (34.2 +/- 17.2 pg/mL to 20.7 +/- 14.0 pg/mL; P = 7.50 x 10(-5)) and of 46.7% after 2 years (40.4 +/- 24.1 pg/mL to 21.5 +/- 23.3 pg/mL; P = 2.48 x 10(-4)) of treatment. Patients with persistent activity of choroidal neovascularization showed a significantly smaller decrease of plasma VEGF levels than patients with dry intervals despite the higher number of injections (P = 0.048). Conclusion: In addition to immediate effects limited to days if not hours, ranibizumab also leads to long-term alterations of systemic VEGF to subnormal levels. Patients with persistent choroidal neovascularization activity showed a less pronounced VEGF decrease. Therefore, VEGF levels might be a useful marker for treatment response