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Nicolas Froger, Valérie Forster, Dorothée Pain, Ivana Ivkovic, Nadège Brunel, Jose Sahel, Serge Picaud; VEGF-R1 activation increases survival of purified retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3385.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) damages are the ultimate and common process characterizing degenerative retinopathies, like glaucoma. In these diseases, no treatment is yet available to directly target RGC degeneration. Previously, we reported that a retinal conditioned medium was able to increase adult RGC survival in pure cultures. Our aim was to therefore identify RGC neuroprotective molecules contained in this medium.
Mixed retinal cell cultures were generated from adult rat retina by enzymatic and mechanical dissociation. After 10-12 days in vitro (DIV) in DMEM medium, the culture medium was changed to neurobasal-A medium (NBA). After 48-hour incubation, the retinal conditioned medium (RCM) was harvested. This medium or control NBA medium were applied on adult rat RGCs purified by immunopanning. Cell survival was assessed by automatic counting of calcein-positive cells on an automatic cell counting platform using the metamorph software.
Application of RCM on pure RGCs produced a long-term survival effect: at 6 days in vitro (DIV) (+191% survival, n=7); 9DIV (+260% survival, n=8), and until 15 DIV (+700% survival, n=6). When this RCM was analyzed by LUMINEX, the presence of VEGF-A was detected at a concentration of 86.8pg/ml (mean±SEM, n=4). To asses if this factor was involved in the RCM-induced survival of RGCs, a VEGF-A antibody was co-applied with RCM. This antibody decreased the RGC survival in RCM by 46% (n=11). The VEGF-induced RGC survival was also observed (+41%, n=19) by adding recombinant VEGF-A (10ng/ml) to the control culture medium. When the VEGF receptor 1 (VEGF-R1) was blocked selectively by the antagonist (ZM323881), the increase in RGC survival induced by recombinant VEGF-A was suppressed. Application of the recombinant VEGF-B, a selective VEGF-R1 agonist, stimulated RGC survival (+31%, n=11). RT-PCR on the freshly purified rat RGCs indicated that RGCs also express the VEGF-R1 receptor in vivo.
These data demonstrated that mixed retinal cells in culture can synthesize and release VEGF-A. They show that this VEGF-A can increase RGC survival by activation of their VEGF-R1. Therefore, our study suggests that this receptor provides an interesting pharmacological target to prevent RGC degeneration in retinal diseases.
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