Martin S. Spitzer

SAFETY PROFILE OF BEVACIZUMAB ON CULTURED HUMAN CORNEAL CELLS

Purpose: To study the corneal biocompatibility of bevacizumab on various cultured human corneal cells. Methods: Cell cultures of corneal keratinocytes (CKs), corneal fibroblasts (CFs), and corneal endothelial cells (CECs) were harvested from human donor eyes and exposed to various concentrations of bevacizumab (0.25-5.0 mg/mL). Cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at days 1 and 4 after exposure. For cytotoxicity testing, confluent cells were cultured in serum-depleted medium, and the MTT test was performed after 24 hours of incubation. Expression of vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR1 and VEGFR2), keratan sulphate (KS), and cytokeratin-3 (AE5) was studied by immunohistochemistry. Live/dead viability/cytotoxicity assay was performed and analyzed by fluorescence microscopy after 24 hours of incubation. Cell morphology was assessed with a phase-contrast microscope after 7 days of exposure with different concentrations of bevacizumab (0.25-5.0 mg/mL), and signs of cellular damage were assessed. Results: No cytotoxic effect of bevacizumab on CKs, CFs, and CECs could be observed when used at a concentration of 5.0 mg/mL or lower. Bevacizumab-treated cells showed no signs of cellular damage compared with the control. CKs, CFs, and CECs stained positively for VEGF, VEGFR1, and VEGFR2. CKs and CECs stained positively for AE5, whereas CFs were immunopositive for KS. Conclusions: Bevacizumab is not toxic to corneal cells of human origin in vitro at doses usually used for treatment of corneal neovascularization, which is 20-fold higher than that used for intravitreal application.

Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells

Aim: To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5), and pig choroidal endothelial cells (CEC). Methods: Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab (0.008–2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5′-bromo-2′-deoxyuridine (BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The “Live/Dead” viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors (VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry. Results: No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity (maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor. Conclusions: These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses (2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium.

Efficacy of intravitreal bevacizumab in treating postoperative pseudophakic cystoid macular edema

PURPOSE: To describe the efficacy of intravitreal bevacizumab (Avastin) in patients with cystoid macular edema (CME) after cataract surgery (Irvine‐Gass syndrome). METHODS: This retrospective case series comprised 16 eyes of 16 patients with CME after cataract surgery refractory to current standard treatment who received an injection of 1.25 mg intravitreal Avastin. The main outcome measures were visual acuity, retinal thickness on optical coherence tomography (OCT), and complications related to treatment. RESULTS: The median duration of CME before treatment with intravitreal Avastin was 14 weeks (range 3 to 84 weeks). Although the mean retinal thickness decreased slightly after intravitreal Avastin, the mean visual acuity remained unchanged. Visual acuity improved by 2 Early Treatment Diabetic Retinopathy Study lines in 1 patient, remained unchanged in 12 patients, and decreased by 2 lines in 2 patients. Repeated Avastin injections did not result in a better outcome. Other than mild ocular irritation, there were no adverse effects of the intravitreal injections. CONCLUSIONS: Intravitreal injection of Avastin, although safe, did not result in improved visual function in patients with postoperative CME. In contrast to findings in a previous case report, the beneficial effect of vascular endothelial growth factor inhibition in Irvine‐Gass syndrome was not observed.

Intraocular lens power calculation and optimized constants for highly myopic eyes

PURPOSE: To determine the accuracy of intraocular lens (IOL) power calculations in eyes with high myopia and to suggest adjusted constants for these cases. SETTING: Centre for Ophthalmology, Eberhard‐Karls‐University, Tuebingen, Germany. METHODS: Patients with high myopia having phacoemulsification with implantation of an AcrySof MA60MA IOL (power range +5.00 to −5.00 diopters [D]) were evaluated. Optical biometry (IOLMaster) and IOL calculations were performed before and after IOL implantation. Because of different optic principal planes of negative‐diopter and positive‐diopter IOLs, separate constants were calculated for these groups. RESULTS: Fifty eyes (32 patients) were evaluated. Thirty eyes (mean AL 31.15 mm ± 1.69 [SD]) had implantation of a positive‐diopter IOL (mean power +3.10 ± 1.50 D) and 18 eyes (mean AL 33.20 ± 2.25 mm), a negative‐diopter IOL (mean power −3.20 ± 1.70 D). Postoperatively, the mean spherical equivalent was −1.42 ± 1.33 D and −0.41 ± 1.81 D, respectively. The difference in optimized constants between positive‐ and negative‐diopter IOLs was significant for all formulas. Power calculation with the SRK II formula showed a wide range of deviation of postoperative refraction from target refraction. Calculation with the Haigis, SRK/T, Holladay 1, and Hoffer Q formulas showed a mean deviation of 0.00 D with an SD of 0.88, 0.92, 1.03, and 1.15, respectively. CONCLUSIONS: Results indicate that the SRK II formula cannot be recommended for IOL power calculation in highly myopic patients. With optimized constants, the SRK/T, Haigis, Hoffer Q, and Holladay 1 formulas produced small deviation of postoperative refraction from target refraction.

Bevacizumab as adjuvant for neovascular glaucoma

Purpose:  We aimed to evaluate the longterm effects of intraocular bevacizumab (Avastin®) injections as adjuvant treatment in patients with neovascular glaucoma.

Z-suture: a new knotless technique for transscleral suture fixation of intraocular implants

The presented Z-suture is a simple, rapid and safe knotless technique that facilitates transscleral suture fixation of various intraocular implants in the ciliary sulcus, such as sutured intraocular lenses, artificial iris prostheses and iris diaphragms. As the knotless approach reliably avoids suture erosion, external fixation can be performed without any protecting scleral flaps or lamellar grooves. The needle is simply passed through the sulcus and the emerging polypropylene suture is secured in the sclera using a zigzag-shaped intrascleral suture (Z-suture). Each pass starts directly adjacent to the exiting site. Five passes are sufficient to reliably fix the suture so that it resists even maximum tractive forces. Once this procedure is done, the suture can be cut without any knot. By avoiding suture knots, and hence the need for intrascleral flaps, this knotless approach may help to reduce suture-related complications such as scleral atrophy, suture erosion and infections.

Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts

Purpose:  To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering.

Comparative toxicity and proliferation testing of aflibercept, bevacizumab and ranibizumab on different ocular cells

Background/aims Vascular endothelial growth factor (VEGF) is a key factor in the pathogenesis of neovascular retinal diseases including age-related macular degeneration. VEGF inhibitors including ranibizumab, pegaptanib or bevacizumab improve retinal morphology and vision in many patients. The recently approved drug aflibercept (VEGF Trap-Eye/Eyelea, Regeneron, Tarrytown, New York, USA) offers a new therapy modality. We therefore tested for toxic and anti-proliferating effects of aflibercept. Methods The effects of aflibercept (0.125, 0.5, 2 mg), ranibizumab (0.125 mg) and bevacizumab (0.3125 mg) after 1, 24, 48 and 72 h on cell morphology via phase contrast pictures, cell viability via MTS assay, total cell amount via crystal violet staining, apoptosis induction via caspase 3/7 assay and proliferation via BrdU assay were investigated. Three ocular cell lines were chosen for toxicology testing: ARPE19 cells, RGC-5 cells and 661W cells. Results Aflibercept did not cause changes in cell morphology, induce apoptosis or cause permanent decrease in cell viability, cell density or proliferation in any cell line or concentration investigated. In general, aflibercept had fewer effects (upregulation or downregulation) compared with controls than bevacizumab or ranibizumab. Conclusions In our experiments, aflibercept did not lead to any negative effects on retinal cell lines and might therefore be used safely in clinical applications.

Ocular surface squamous neoplasia as the first apparent manifestation of HIV infection in Malawi

Purpose:  To evaluate the prevalence of undiagnosed and asymptomatic HIV infection in patients with ocular surface squamous neoplasia (OSSN) in an urban patient population in Malawi.

Decellularized Bovine Corneal Posterior Lamellae as Carrier Matrix for Cultivated Human Corneal Endothelial Cells

Purpose: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs). Methods: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet’s membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), natrium hydrogen carboanhydrase (Na+/HCO3−), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry. Results: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet’s membrane. A significant DNA reduction (mean before decelluraziation 365.3 ± 88.6 ng/mg, mean after decelluarization 23.2 ± 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 ± 179 cells/mm2 on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na+/HCO3−, and Na+/K+-ATPase. Conclusions: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.