Rajesh K. Sharma

Intracameral bevacizumab effectively reduces aqueous vascular endothelial growth factor concentrations in neovascular glaucoma

Vascular endothelial growth factor (VEGF) concentrations in both the aqueous and vitreous are raised in ocular ischaemia secondary to exudative age-related macular degeneration, diabetes mellitus and other retinal vascular diseases.1 2 Retinal ischaemia upregulates VEGF production, resulting in neovascularisation of the retina and the iris (NVI). VEGF is significantly increased in neovascular glaucoma (NVG).3 In the following case series, we show increased aqueous VEGF concentrations in two patients with NVI, which significantly declined, with concurrent resolution of NVI, after off-label intracameral injections of bevacizumab. Two patients with NVI secondary to proliferative diabetic retinopathy were included in the study. After informed consent had been obtained, according to a protocol approved by the University of Florida Institutional Review Board, aqueous was collected before the injection of 0.05 ml intracameral bevacizumab (25 mg/ml) and …

Evaluation of cytotoxic effects of bevacizumab on human corneal cells.

Purpose: Corneal neovascularization contributes to corneal opacification in inflammatory conditions of the cornea and severely compromises the success of corneal transplantation. Vascular endothelial growth factor (VEGF) plays an important role in stimulating and maintaining corneal neovascularization. Anti-VEGF therapy, especially the use of anti-VEGF antibody bevacizumab, has gained popularity in the management of retinal neovascularization and is being used topically for corneal neovascularization. The aim of this study was to investigate the safety profile of bevacizumab on human corneal cell lines. Methods: Human corneal epithelial and fibroblast cell lines and an umbilical vascular endothelial cell line were treated with increasing doses of bevacizumab. The effect of this treatment on cell viability was assessed by WST-1 and crystal violet staining assays. Cytotoxicity was also assessed by fluorescent microscopy and flow cytometric evaluation of propidium iodide-stained cells. Results: In the cytotoxicity experiments, there was no difference in cell numbers after 24-hour exposure compared with control in any of the cell lines at the concentrations tested (P > 0.05 to 0.98). Conclusion: Bevacizumab was nontoxic to human corneal epithelial and fibroblast cells at 3 different concentrations.

Evaluation of Differential Toxicity of Varying Doses of Bevacizumab on Retinal Ganglion Cells, Retinal Pigment Epithelial Cells, and Vascular Endothelial Growth Factor–Enriched Choroidal Endothelial Cells

PURPOSE To evaluate in vitro the effects of bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, on retinal pigment epithelial cells (RPE) and retinal ganglion cells (RGC), at doses that were inhibitory to VEGF-enriched choroidal endothelial cells (CEC). METHODS Monkey CEC (RF6A), human RPE cells (ARPE-19), and rat RGC (RGC-5) were exposed for 24 h to increasing doses of bevacizumab. Cell numbers were quantified with WST-1 assay. Cell death was assessed using propidium iodide (PI) staining via flow cytometry and fluorescent microscopy. RESULTS Bevacizumab was inhibitory to RF6A at 2.0 mg/mL (P < 0.005). No effect on cell viability was noted on ARPE-19 and RGC-5 cell lines at this particular dose of bevacizumab. These results were supported by fluorescent microscopy of PI-stained cells. CONCLUSIONS VEGF-stimulated proliferation of CEC was inhibited by bevacizumab. Bevacizumab was not cytotoxic to human RPE and rat RGC in vitro at a dose that is inhibitory to monkey CEC.

In vitro evaluation of bevacizumab toxicity on a retinal ganglion cell line

Purpose:  The effects of bevacizumab on cell viability and proliferation in a commonly used retinal ganglion cell line, RGC‐5, were examined.

Preconditioning protects the retinal pigment epithelium cells from oxidative stress‐induced cell death

Purpose:  The cytotoxic effects of oxidative stress, which play an important role in ocular diseases, are well known. In this study, we investigated the effect of non‐lethal doses of oxidative stress on various cell functions, namely cell viability, cell attachment and cell migration in a widely used retinal pigment epithelium (RPE) cell line (ARPE‐19).

Validation of Molecular and Genomic Biomarkers of Retinal Drug Efficacy: Use of Ocular Fluid Sampling to Evaluate VEGF

The use of tissue- and cell-based methods in developing drugs for retinal diseases is inefficient. Consequently, many aspects of ocular drug therapy for retinal diseases are poorly understood. Biomarkers as prognostic indicators of change are needed to optimize the use of drugs. VEGF is considered an important target of drug therapy and VEGF levels in tissue are indicative of solid tumor growth. However, since many aspects of VEGF as a biomarker of ocular disease have not been validated, it has been difficult to ascertain without invasive procedures whether VEGF in the eye is a biomarker of response to drug therapy. Using published papers, registered clinical trials, and proteomic databases we assessed the earlier evidence for VEGF as an exploratory biomarker of proliferative and vasculopathic disease of the retina and asked whether the molecule has been rigorously validated in clinical trials. The emerging use of aqueous humor sampling has made it possible to explore biomarkers in oculo, and determine whether they are predictive of drug efficacy. We present data supporting the use of aqueous humor to validate drug-signaling pathways and biomarkers in the eye. In addition, we recommend convening a collaborative congress to help standardize the identification, validation, and use of biomarkers in retinal disease.

Limitations in assessing nerve growth factor levels in aqueous humor samples from human eyes

BackgroundNerve growth factor (NGF) helps in the healing and survival of ganglion cells, photoreceptors, and optic nerve after injury and has been implicated to have a role in pathophysiology of glaucoma. So far, in animal studies, injury to iris in vitro has revealed an increase in NGF levels in aqueous. There is a great interest in investigating the levels of NGF in human aqueous in glaucomatous eyes, as suggested by animal studies, to gain a better understanding of the pathophysiology of glaucoma.FindingsIn this study, we examined the presence of NGF levels in aqueous humor collected from human eyes and the limitations in determining the NGF levels in human samples. NGF was assessed by ELISA immunoassay in undiluted aqueous samples collected from 32 consecutive patients undergoing surgery for cataract (control) or primary open angle glaucoma (POAG). Recombinant NGF was used as positive control. NGF levels were below undetectable levels in aqueous humor from eyes with POAG and controls by immunoassay. Less than 10% of samples had detectable NGF levels and these were considered outliers.ConclusionOur result highlights the undetectable levels of NGF in human aqueous samples.