Functional Analysis of Semaphorin 3A in Retinal Ganglion Cells under Hypoxia In Vitro

Glaucoma is a progressive neurological disease and a main cause of blindness worldwide. Glaucoma causes irreversible damage to retinal ganglion cells (RGCs) and axons, leading to impaired visual function if not properly treated. Currently, there is no cure for chronic glaucoma, and the main therapeutic goal is to maintain visual function by preventing further disease progression. Thus, neuroprotective strategies are necessary to prevent the progression of optic neuropathy in glaucoma. We first analyzed from the viewpoint of hypoxic stimulation because that circulatory failure may occur even in anterior glaucoma before visual field abnormalities appear at recent glaucoma symptoms. Here, we examined the role in RGCs of Semaphorin 3A, which plays an important role in determining axon elongation during development. Materials and Methods: RGC cultures were prepared from the retinas of 4-5-day-old CrIJ Wistar rats and subjected to hypoxia stress (1% O-2) or normoxia (21% O-2). The expression of class 3 Semaphorins was examined by reverse transcription polymerase chain reaction, western blot, and immunofluorescence analyses. Results: Among the secretory class 3 Semaphorins, only the expression of Semaphorin 3A was increased in RGCs under hypoxia conditions; there was no change in the expression of other Semaphorins (3B, 3C, and 3F). Western blot and immunofluorescence analyses revealed down-regulation of the RGC differentiation marker TUJ1 under hypoxia. Hypoxia-induced down-regulation of TUJ1 in RGCs was blocked by siRNA-mediated knockdown of Semaphorin 3A. A multi-array cytokine assay revealed that vascular endothelial growth factor expression was increased in RGCs under hypoxic conditions; its expression was suppressed by siRNA knockdown of Semaphorin 3A. Conclusions: These results indicate that Semaphorin 3A exerts an important neuroprotective function that is closely related to interactions with vascular endothelial growth factor.