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

Abstract

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.

• Authors: Di Cao, Jing Li, Xiao Wang, Jing Wang, Ruyuan Liu, Jing Lu, Qiuhui Liu, Yan Luo
• Journal: Experimental eye research (Exp Eye Res) Vol. 213 Pg. 108836 (12 2021) ISSN: 1096-0007 [Electronic] England
• PMID: 34774487 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2021. Published by Elsevier Ltd.

Chemical References

• Claudin-3
• Cldn3 protein, mouse
• Proto-Oncogene Proteins c-bcl-2
• Vascular Endothelial Growth Factor A
• vascular endothelial growth factor A, mouse

Topics

• Animals
• Animals, Newborn
• Apoptosis
• Blotting, Western
• Claudin-3 (metabolism)
• Dependovirus (genetics)
• Down-Regulation (physiology)
• In Situ Nick-End Labeling
• Mice
• Mice, Inbred C57BL
• Microscopy, Fluorescence
• Neovascularization, Physiologic (physiology)
• Proto-Oncogene Proteins c-bcl-2 (metabolism)
• Real-Time Polymerase Chain Reaction
• Retina (metabolism)
• Retinal Ganglion Cells (pathology)
• Retinal Vessels (physiology)
• Vascular Endothelial Growth Factor A (metabolism)