Levels of vascular endothelial growth factor-A(165b) (VEGF-A(165b)) are elevated in experimental glaucoma

PURPOSE: Although ischemia has previously been suggested to contribute to the pathogenesis of glaucoma, neovascularization is not implicated in glaucoma. Because vascular endothelial growth factor-A (VEGF-A) is a key mediator in neovascularization response, we investigated the levels of the major pro-angiogenic (VEGF-A(164)) and anti-angiogenic VEGF-A subtypes (VEGF-A(165b)) in the retina during experimental glaucoma. METHODS: Glaucoma was induced unilaterally in rats by injecting 1.9 M hypertonic saline solution in the episcleral veins. The contralateral eye served as the control. The intraocular pressure (IOP) of each eye was measured via Tonopen in conscious rats. Eyes were enucleated either on the 5th or the 10th day of elevated IOP. Whole retinal lysates were separated by SDS–PAGE and transferred to PVDF membranes. Levels of VEGF-A(164) and VEGF-A(165b) were analyzed by western blotting using specific antibodies. In a different group of rats, retinal ganglion cells were retrogradely labeled by injecting Fluorogold in the superior colliculus a week before the induction of glaucoma. After the eyes were enucleated on the fifth day of elevated IOP, posterior eye cups were sectioned using a cryostat. Levels and localization of VEGF-A(164) and VEGF-A(165b) were examined in retinal sections by immunohistochemistry. RESULTS: VEGF-A(164) levels remained unchanged between the control and glaucomatous retinas after five days (p=0.341) and 10 days of elevated IOP (p=0.117). The presence of the anti-angiogenic VEGF-A isoform has not been previously reported in the rat. An antibody specific to VEGF-A(165b) detected the anti-angiogenic protein in the rat retina. VEGF-A(165b) levels were significantly increased (2.33±0.44 fold, p=0.014) in the glaucomatous retinas compared to those in controls after five days of elevated IOP. VEGF-A(165b) levels were not different (p=0.864) between the control and glaucomatous retinas following 10 days of elevated IOP. Expression of both VEGF-A(164) and VEGF-A(165b) were observed in the retinal ganglion cells (RGC) and inner nuclear layer (INL). CONCLUSIONS: Five day elevation of IOP leads to an increase in the anti-angiogenic VEGF-A(165b) levels but not in the pro-angiogenic VEGF-A(164) levels in the glaucomatous retina. VEGF-A(165b) levels return to baseline after 10 days of elevated IOP, and VEGF-A(164) levels remain unchanged. We speculate that the short-term elevation of VEGF-A(165b) levels and/or the unchanged levels of VEGF-A(164) contribute to the lack of neovascularization in the glaucomatous retina.