Abstract
Purpose :
Retinal angiogenesis starts with the radial migration of astrocytes from the optic nerve to the retinal periphery. Subsequently, astrocytes secrete molecular factors such as vascular endothelial growth factor (VEGF) to recruit new blood vessels from the optic disc. Successively, these form along the VEGF gradient the superficial vascular plexus towards the retinal periphery. Afterwards they spread deeper to form the intermediate and deep retinal vascular plexus. Here, we characterized the impact of VEGF signaling on developmental retinal angiogenesis and astrocyte recruitment. To this end, we generated and analyzed a mouse model with tamoxifen-inducible deletion of vascular endothelial growth factor 2 (Vegfr2) in the entire eye.
Methods :
Neonatal mice homozygous for a floxed Vegfr2 allele and hemizygous for the CAG-CreER transgene were treated with tamoxifen eye drops to induce Vegfr2 deletion in the eye. Retinal structure was studied by light microscopy, immunohistochemistry and FITC-dextran perfusions. Murine optic nerve astrocytes (MONA) were isolated and VEGFA-dependent proliferation was determined using the crystal violet assay. QPCR and western blotting were performed to analyze the molecular mechanisms.
Results :
Lack of postnatal VEGF signaling (Vegfr2-/-;CAG-Cre = Vegfr2Δeye) resulted in persistence of the hyaloid vasculature concomitant with an avascular retina at postnatal day 12. Semithin retinal sections showed a regular retinal layering, but a remarkably thickened nerve fiber layer. Quantification of glial fibrillary acidic protein (GFAP)-stained retinal sections showed a significantly higher number of astrocytes in Vegfr2Δeye retinae. Western blot analyses using hypoxia inducible factor 1 α (HIF1α) antibody revealed HIF1α stabilization in Vegfr2Δeye retinae concomitant with significantly increased VEGFA levels, thus indicating hypoxia. VEGFA-stimulated MONA showed a significantly higher proliferation capacity in vitro.
Conclusions :
VEGF signaling is critically required for retinal vascular development. Its deletion results in an avascular retina and an increased number of astrocytes. Further in vitro experiments revealed that MONA proliferate upon VEGFA stimulation. In summary, our data indicate that VEGF signaling not only influences retinal angiogenesis, but also homeostasis of retinal astrocytes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.