Avastin increases RPE cell death under oxidative stress Avastin (bevacizumab) decreases survival of cultured retinal pigment epithelial cells stressed by hydrogen peroxide, according to a new study.

Vascular endothelial cell growth factor (VEGF) is strongly induced by oxidative stress in retinal pigment epithelial (RPE) cells, and VEGF-A is a survival factor for various cell types. VEGF-A expression is increased in the RPE cells of the macula in patients with age-related macular degeneration (AMD), a condition associated with a high risk of choroidal neovascularization (CNV). The presumed principal source of VEGF-A in exudative AMD is the RPE, and oxidants have been reported to increase the deposition of oxidized proteins or other oxidized compounds in Bruch's membrane, in a process that may involve complement activation and inflammation, provoking proangiogenic VEGF-A release from the RPE in patients with exudative AMD. In addition, oxidant compounds, per se, have been shown to stimulate VEGF-A release from the RPE.

Cellular damage resulting from oxidative stress in RPE cells and photoreceptors may play a causative role in aging of the RPE. Oxidative stress-induced RPE cell apoptosis has been proposed as a major pathophysiological mechanism of AMD. In particular, RPE cell apoptosis is an important feature of the advanced form of dry AMD. Thus, oxidative stress induces VEGF-A expression from the RPE and also RPE death, suggesting a role for such stress in both neovascular and advanced dry AMD.

Although current treatments that target VEGF-A have demonstrated the best clinical outcomes of all approaches trailed to date, concern about broad inhibition of VEGF-A activity in AMD eyes remains. Inhibition of VEGF-A has been reported to lead to geographic atrophy and poor visual outcome in some patients with neovascular AMD. Also, RPE tears and choroidal atrophy in specimens from patients with treated AMD raise questions about the long-term safety of anti-VEGF-A treatment.

Methods and Results

ARPE-19 cells were treated with hydrogen peroxide, and cell death was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Survival analysis was performed with pretreatment of VEGF-A–neutralizing antibodies (Avastin), VEGF receptor tyrosine kinase inhibitor (SU5416), or VEGF-A receptor-neutralizing antibodies (anti-VEGF-R1 and anti-VEGF-R2). The expression of VEGF-A, -R1, -R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR or Western blot analysis. Phosphorylation of VEGF-R2 was detected with immunoprecipitation and immunoblot analysis.

Hydrogen peroxide–induced cell death was promoted by pretreatment with VEGF-A and anti-VEGF-R2–neutralizing antibodies, but not with anti-VEGF-R1–neutralizing antibody (FIGURE). Phosphorylation of VEGF-R2 in RPE cells was induced by hydrogen peroxide, and pretreatment with anti-VEGF-A–neutralizing antibody inhibited phosphorylation. Phosphorylation of Akt under oxidative stress was abrogated by pretreatment with neutralizing antibodies against either VEGF-A or SU5416.

Discussion and Conclusions

The results of this study imply that neutralization of VEGF-A signaling with an anti-VEGF-A agent in AMD eyes influences RPE cell survival, which is essential for visual recovery and reduction of AMD recurrence. It may therefore be important to modulate the extent of VEGF-A blockade, or to specifically and selectively inhibit only one or a few of the angiogenic actions of VEGF-A, when considering VEGF-A inhibition as a treatment strategy.

Avastin is a monoclonal antibody binding to all VEGF-A isoforms. Experimental investigations in rats, rabbits, and primates showed that intravitreal bevacizumab at a different concentration did not cause any functional and morphologic retinal toxicity. In vitro cellular assays examining exposure to Avastin have shown little toxic effect on ganglion cells, neuroretinal cells, RPE cells, choroidal endothelial cells, and corneal epithelial cells. However, this study found high doses of Avastin significantly induced RPE cell death under conditions of higher oxidative stress, which may be attributable to blocking of the VEGF-A autocrine survival signal. However, the dose of Avastin used in this study was greater than is used clinically, and RPE cell death was induced only at higher levels of oxidative stress. Further clinical evaluation of the long-term safety of Avastin is needed.

The present study provides evidence that VEGF-A assists in RPE cell survival when cells are exposed to oxidative stress. The results imply that neutralization of VEGF-A signaling with an anti-VEGF-A agent, such as Avastin, in AMD eyes influences RPE cell survival. A high level of VEGF-A secreted from RPE cells under oxidative stress conditions may participate in the pathogenesis of exudative AMD (by stimulating CNV); however, VEGF-A may have a beneficial effect in assisting RPE cell resistance against oxidative stress. Avastin, now extensively used in the ophthalmic field, may also affect RPE cell survival under conditions of high oxidative stress. Thus, the extent or specificity of VEGF-A blockade, and the level of oxidative stress, may affect treatment outcomes (survival of RPE cells, restoration of outer blood-retinal barrier, or geographic atrophy) when anti-VEGF-A treatment is used in patients with neovascular AMD.

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Invest Ophthalmol Vis Sci. 2010 Feb;51(2):1190-7