The effect of AAV-mediated downregulation of Claudin-3 on the development of mouse retinal vasculature.

Retinal vascular development is a very tightly regulated and organized process of vessel formation and regression to generate the mature vasculature system. Claudin-3 has been found to be required for the normal development of the neural retina and its vessels in zebrafish in our recent study. In this study, we investigated whether Claudin-3 played a role in the development of mouse retinal vasculature. Immunofluorescent staining was performed to detect the expression and localization of Claudin-3 in the mouse retina. Intravitreal injection of a recombinant adeno-associated virus (AAV) expressing a short hairpin RNA targeting Claudin-3 mRNA was performed to down-regulate Claudin-3 expression in retina in neonatal (Postnatal Day 3, P3) C57BL/6J mice. Retinal vessels were examined by isolectin B4 immunofluorescent staining on the whole-mount retinas and frozen retinal sections at P10. The apoptotic retinal ganglion cells (RGCs) were measured by TdT-mediated dUTP nick-end labelling (TUNEL) staining. Vascular endothelial growth factor A (VEGF-A) expression was detected by immunofluorescent staining. The protein levels of Claudin-3, VEGF-A and B cell lymphoma 2 (Bcl-2) were evaluated by Western blot at P7, P10 and P14. We found that Claudin-3 mainly expressed in the RGCs and progressively increased during the retinal development. The AAV-mediated downregulation of Claudin-3 at P3 impeded the development of retinal deep vascularization of P10 mouse, but without effect on the development of the retinal superficial plexus. Claudin-3 knockdown increased RGC apoptosis and reduced the expression of VEGF-A and Bcl-2 in the retinas. These results suggested that the downregulation of Claudin-3 induced RGC apoptosis and impeded the mouse retinal vascular development by downregulating the levels of VEGF-A and Bcl-2.