MDM2/P53 perturbation in endothelial cells causes blood-retina barrier defects that are restored by activating beta-catenin-dependent signaling

Abstract

Purpose : The blood-CNS barriers protect the CNS and their dysfunction is associated with disease, e.g., in diabetes mellitus. Reduction of P53 alleviates effects of diabetes on non-CNS vasculatures in vivo. We seek to determine the roles of mouse double minute 2 homologue (MDM2) and P53 in blood-retina barrier function and the interactions with beta-catenin (CTNNB1) dependent (“canonical”) signaling.

Methods : We performed RNAseq on human retinal microvascular ECs (HRMVECs) and activated P53 and/or frizzled4 (FZD4) using nutlin-3 and the FZD4-ligand norrin (gene symbol NDP). siRNA or overexpression was used to investigate the function of differentially expressed genes in HRMVECs and the bEnd.3 brain EC-line. Mice with EC-specific Mdm2 deletion, Mdm2;Trp53 compound deletion, or EC-specific Mdm2 deletion with activation of constitutively active beta-catenin were analyzed for barrier defects and inflammation markers.

Results : In HRMVECs, nutlin-3 induced inflammation-linked genes including ICAM-1, CCL2, and CXCL1. Furthermore, nultin-3 strongly inhibited NDP-signaling in a P53-dependent manner and altered the expression of genes potentially involved in NDP-signaling including NCAPH and DRAXIN. SiRNA against condensin complex subunit Ncaph was sufficient to strongly inhibit proliferation and norrin signaling, whereas DRAXIN had no effect on norrin signaling. Short-term EC-specific Mdm2 ablation resulted in increased P53 immunostaining in retinal ECs in the absence of apoptosis. EC-specific Mdm2 recombination in mature mice with quiescent vasculature caused BRB defects, which were associated with strong inflammation and only minor reduction in wnt signaling, highlighting potentially different responses of proliferating vs. quiescent CNS ECs. Retinal vascular inflammation was suppressed in compound mutant Mdm2;Trp53 EC-specific KO mice. Constitutive activation of beta-catenin signaling in mature Mdm2 ECKO ECs alleviated BRB defects.

Conclusions : This study identifies novel roles of MDM2/P53 in control of the BRB and highlights that activation of beta-catenin-dependent signaling in ECs can alleviate barrier defects of different etiologies.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.