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Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn

Purpose:  That vascular endothelial growth factor (VEGF) plays a major role in inflammatory angiogenesis has been well established. This pilot study was designed to evaluate experimental treatment with bevacizumab eyedrops in corneal neovascularization induced by alkali burn. The feasibility of topical administration, corneal cell viability and corneal penetration were investigated in an animal model.

Vitreous Levels of Bevacizumab and Vascular Endothelial Growth Factor-A in Patients with Choroidal Neovascularization

PURPOSE To investigate the vitreous levels of bevacizumab and vascular endothelial growth factor-A (VEGF-A) after intravitreal injection of the drug in patients with choroidal neovascularization (CNV). DESIGN Interventional case series. PARTICIPANTS Eleven eyes of 11 patients with submacular hemorrhage and CNV due to age-related macular degeneration (n = 10) or angioid streaks (n = 1). METHODS All patients were treatment naïve except for a single dose of intravitreal injection of bevacizumab (1.25 mg/50 muL dose) and subsequent vitrectomy after various intervals (1-101 days) because of active and progressive lesion. Intravitreal free bevacizumab and VEGF-A levels were measured using enzyme-linked immunosorbent assay and microsphere-based immunoassay, respectively. Vitreous VEGF-A isoforms were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. MAIN OUTCOME MEASURES Intravitreal bevacizumab and VEGF-A levels were measured and pharmacokinetic parameters were calculated. RESULTS Pharmacokinetics of intravitreal bevacizumab followed a 2-compartment model with initial and terminal half-lives of 0.5 and 6.7 days, respectively. Bevacizumab could be detected in all cases, ranging from 2.63 ng/ml to 165 microg/ml. The peak concentration was observed on the second day after intravitreal bevacizumab injection. Vitreous free VEGF-A levels ranged from 0.2 to 33.9 pg/ml and showed a negative correlation with the bevacizumab concentration (P<0.001; r = -0.955) and a positive correlation with time (P<0.001; r = 0.964). However, the percentage expression of VEGF-A(165) exhibited a positive correlation with the bevacizumab concentration (P = 0.032, r = 0.645) and a negative correlation with time (P = 0.007, r = -0.755). A time-dependent increase was found for the percentage expression of VEGF-A(189) (P = 0.023, r = 0.673). Neither bevacizumab- nor time-related alterations were found for VEGF-A(121). CONCLUSIONS Based on pharmacokinetics, the interval of 6-7 weeks would be appropriate for efficacy, although clinical trials should guide dosing recommendations. Vitreous levels of free VEGF-A showed a negative correlation with the bevacizumab concentration, which confirmed the in vivo binding affinity of bevacizumab to VEGF-A. The analysis of the VEGF-A isoforms suggests differences of interaction between bevacizumab and individual VEGF-A isoforms.

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.

Effect of bevacizumab on inflammation and proliferation in human choroidal neovascularization.

OBJECTIVE To evaluate the effect of bevacizumab (Avastin; Genentech, Inc, South San Francisco, California) on inflammation and proliferation in human choroidal neovascularization (CNV) secondary to age-related macular degeneration. METHODS Retrospective review of interventional series of 38 patients who underwent choroidal neaovascular membrane (CNVM) extraction. Twenty-four patients received intravitreal bevacizumab 1 to 154 days preoperatively (bevacizumab CNV group). Fourteen patients received no preoperative therapy (control CNV group). The CNVM were stained for cytokeratin 18, CD68, CD45, intercellular adhesion molecule (ICAM)-1, E-selectin, Ki-67, Thy-1, and endostatin. RESULTS No significant difference was detected in ICAM-1 and E-selectin expression between groups. The density of leukocytes in the bevacizumab CNV group (median, 271.61 cells/mm(2)) was higher than in the control CNV group (median, 116.87 cells/mm(2); P = .07), but without significance. Density of macrophages (median, 4661.95 cells/mm(2)), proliferative activity (median, 160.19 cells/mm(2)), and percentage of Thy-1-expressing vessels (median, 100%) were significantly higher in the bevacizumab CNV group than in the control CNV group (median, 882.66 cells/mm(2), P < .001; median, 34.34 cells/mm(2), P < .001; and median, 80%, P < .001, respectively). Endostatin immunoreactivity was considerably stronger in the retina pigment epithelium (RPE)-Bruch membrane complex (median, 3; range, 2-3; P < .001), and stroma (median, 3; range, 1-3; P < .001) of the bevacizumab CNV group than control CNV group (median, 1.5; range, 0-3 and median, 1; range, 0-3, respectively). CONCLUSIONS Unexpectedly, CNVM from patients treated by bevacizumab are characterized by significantly high inflammatory and proliferative activity and enhanced endostatin expression. These characteristics need to be considered when protocols for combination therapies are established.

Influence of verteporfin photodynamic therapy on inflammation in human choroidal neovascular membranes secondary to age-related macular degeneration.

Purpose: To examine the short- and long-term consequences of verteporfin photodynamic therapy (PDT) on inflammation with regard to infiltration of macrophages and leukocytes and expression of thy-1 in human choroidal neovascularization membranes (CNV) secondary to age-related macular degeneration (AMD). Methods: Retrospective review of an interventional case series of 43 patients who underwent removal of CNV. Twenty patients were treated with PDT 3 to 246 days preoperatively. Twenty-three CNV without previous treatment were used as control. CNV were stained for CD34, CD105, cytokeratin18, Ki-67, thy-1, an endothelial cell glycoprotein known to be upregulated only by inflammatory cytokines, CD68 (macrophages), and CD45 (common leukocyte antigen). Results: Specimens treated by PDT 3 days previously showed significantly reduced endothelial thy-1 expression (P = 0.008), leukocyte (P = 0.04) and macrophage (P = 0.0063) infiltration, and proliferative activity (P = 0.02) compared to control CNV. Specimens at longer intervals after PDT, in contrast, disclosed a significantly increased expression of thy-1 (P = 0.004), infiltration with leukocytes (P = 0.044) and macrophages (P = 0.01), and proliferative activity (P = 0.03) compared to CNV excised 3 days after PDT. Conclusions: The rebound effect after PDT seems to be based on an inflammatory response that contributes to enhanced proliferation. These data support the need for an anti-inflammatory therapy as adjuvant to PDT.

Verteporfin photodynamic therapy induced apoptosis in choroidal neovascular membranes

Aim: To evaluate the impact of verteporfin photodynamic therapy (PDT) on the induction of apoptosis in choroidal neovascular membranes (CNV) secondary to age related macular degeneration. Methods: Retrospective review of 22 surgically excised CNV. 12 of these patients had been treated with PDT 3–146 days previously. Apoptotic cells were detected with the TUNEL technique and compared to the expression of CD34 (endothelial cells, EC), CD105 (activated endothelial cells), Ki-67 (proliferation marker), and cytokeratin18 (retinal pigment epithelial cells, RPE). Results: CNV excised 3 days after PDT were characterised both by collapsed and patent vessels. The EC displayed a statistical significant positive TUNEL reaction when compared to the remaining treated CNV (p<0.001) and untreated CNV (P = 0.002). The proliferative activity was reduced. CNV excised 1–5 months after PDT displayed a patent vascularisation and high proliferative activity. All membranes either treated or untreated disclosed only sporadic TUNEL positive cells within the stroma and the RPE. Conclusions: Verteporfin PDT leads to selective and effective damage of EC within CNV. Both patent and occluded vessels were lined by apoptotic EC. This finding and the increased expression of proliferation marker at later time points suggest that revascularisation after PDT is caused by angiogenesis rather than recanalisation.

Human anterior lens capsule as carrier matrix for cultivated human corneal endothelial cells.

Purpose: To evaluate the potential of human anterior lens capsule (HALC) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs). Methods: HALCs obtained from 12 patients during cataract surgery were exposed to enzyme digestion to dissolve the lens epithelium and were plated with the epithelial side up in 6-well plates. HCECs were harvested from human donor eyes and seeded on HALCs. Cell morphology was assessed with a phase-contrast microscope after 6 hours of incubation and at days 1, 3, 5, and 7. The number of HCECs counted at each capsule was pooled, and the density was expressed as cells per square millimeter after 7 days in culture. Live/Dead viability/cytotoxicity assay was also performed and analyzed by fluorescence microscopy after 7 days of incubation during confluence. Expression of zonula occludens-1, connexin-43, Na+/K+-adenosine triphosphatase, and cytokeratin-3 (AE5) were investigated by immunohistochemistry. Results: Seven days after seeding on HALCs, the HCECs were grown to confluence and formed a continuous viable monolayer with a mean cell density of 3012 ± 109 cells per square millimeter, which mimics native corneal endothelial cells. Immunohistochemistry analysis demonstrated strongly positive staining for AE5, zonula occludens-1, connexin-43, and Na+/K+-adenosine triphosphatase. Conclusions: Cell density and morphology of HCECs on HALCs were similar to those of healthy corneas. Phenotypical properties of HCECs on HALCs imply that the HCEC sheets are capable of maintaining intact barrier and ionic pump functions in vitro. HALCs might, therefore, be recommended as a potential scaffold for ex vivo expansion of HCECs, possibly providing an autologous biologic substrate for therapy of isolated corneal endothelial diseases.

Matrix metalloproteinases in human choroidal neovascular membranes excised following verteporfin photodynamic therapy

Aim: To evaluate expression of proangiogenic matrix metalloproteinases (MMP) 2 and 9 at distinct intervals after verteporfin photodynamic therapy (PDT) in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD). Methods: Retrospective review of an interventional case series of 49 patients who underwent removal of CNV. Twenty-six patients were treated with PDT 3 to 383 days prior to surgery. Twenty-three CNV without previous treatment were used as controls. CNV were stained for CD34, cytokeratin 18, endostatin, MMP-2 and MMP-9 by immunohistochemistry. Results: CNV without previous therapy disclosed MMP-2, MMP-9 in RPE–Bruch’s membrane, vessels and stroma in different intensities. Three days after PDT, MMP-9 expression was significantly weaker in stroma (p = 0.0019). Endostatin was significantly reduced in vessels (p<0.001). At longer post-PDT intervals, a significant increase of MMP-9 in stroma (p = 0.037) and of endostatin in RPE–Bruch’s membrane (p = 0.02), vessels (p = 0.005) and stroma (p<0.001) were disclosed. No significant changes in MMP-2 expression were detected. Conclusions: PDT induced an early, temporary decrease in MMP-9 and endostatin expression. At longer intervals, MMP-9 increase is possibly associated with the angiogenic process responsible for recurrence after PDT. MMP-9, however, acts as a double-edged sword by concomitant induction of endostatin, an endogenous inhibitor of angiogenesis.

Toxic Effects of Recombinant Tissue Plasminogen Activator on Cultured Human Corneal Endothelial Cells

PURPOSE Human corneal endothelial cells (HCECs) are nonmitotic cells. Any intracameral application of a drug requires evaluation of the potential apoptotic and toxic effects. Intracameral recombinant tissue plasminogen activator (rtPA) is successfully used for the treatment of severe and prolonged postoperative fibrin reaction. This study was undertaken to investigate the toxicity of rtPA on cultured HCECs to determine its safety for clinical use. METHODS Cell cultures of HCECs were harvested from human donor eyes and exposed to various concentrations of rtPA (10-200 microg/mL). For cytotoxicity testing, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and the live/dead viability/cytotoxicity assay were performed. Annexin V binding combined with propidium iodide (PI) costaining was used for the distinction of viable, early, and late apoptotic cells. Odds ratios (ORs) and confidence intervals (CIs) were calculated for 50 microg/mL, 100 microg/mL, and 200 microg/mL. Cell morphology was studied after 24 hours of exposure to rtPA to identify cellular damage. Immunolocalization of zonula occludens 1 (ZO1) was performed to analyze intercellular barrier disturbance in the presence of rtPA. RESULTS Reduction of mitochondrial dehydrogenase activity after rtPA exposure was dose dependent and suggested comparable toxicity with the data obtained from the live/dead assay. The mean number of Annexin V-FITC and PI-positive cells was not significantly increased at concentrations of 50 microg/mL and 100 microg/mL. At 200 microg/mL, however, the ORs were 5.082 +/- 0.213 (95% CI, 3.962-6.203; P < 0.001) for apoptosis and 6.154 +/- 0.196 (95% CI, 5.123-7.181; P < 0.001) for necrosis. In addition, increasing concentrations of rtPA resulted in a fading immunopositive staining for ZO1. CONCLUSIONS These data suggest a dose-dependent toxic effect of rtPA on HCECs in vitro. Although intracameral rtPA concentrations up to 100 mug/mL seem to be clinically safe, the use of concentrations higher than 125 microg/mL might induce irreversible cell death and should be restricted to selected cases.

Long-term visual acuity and its predictors after cataract surgery in patients with uveitis.

Purpose. To analyze the outcomes of phacoemulsification and posterior intraocular lens (IOL) implantation in patients with uveitis and to determine factors responsible for poor visual outcome. Methods. The records of 155 patients (180 eyes) with uveitis who had phacoemulsification and IOL implantation between August 2001 and March 2008 were examined retrospectively. Best-corrected visual acuity (BCVA) was recorded at the immediate preoperative visit and at follow-up examinations every 3 months. At each postoperative visit, a complete ophthalmologic examination was performed. The postoperative visual outcomes and complications were analyzed. Univariate regression analysis was done to determine risk factors for poor visual acuity during follow-up. Results. The mean follow-up was 31.4 months (range 3–78 months). An underlying systemic disease was present in 70 (45.2%) patients (82 eyes, 45.6%). The mean preoperative logMAR BCVA was 1.13±0.62 (95% CI: 0.85–1.02) and increased to 0.42±0.57 (95% CI: 0.32–0.59) at last medical visit (p<0.001). A total of 107 eyes (59.4%) had postoperative complications including posterior capsular opacification, newly developed macular edema, recurrence of uveitis, macular epiretinal membrane, and deposits on the IOL surface. Preoperatively observed macular lesions was the factor most strongly associated with poor visual outcome after cataract surgery (odds ratio: 5.43; 95% CI: 3.41–7.34; p<0.001). Anterior segment pathologies, age at surgery, etiology of uveitis (idiopathic, uveitis associated systemic disease), and gender did not influence visual rehabilitation after surgery (p>0.05). Conclusions. The outcomes of phacoemulsification and IOL implantation in patients with uveitis were satisfactory. Patients with observed preoperative macular lesions are at risk for poor visual outcome.