The Protective Role of Endothelial RIPK3 in Retinopathy of Prematurity

Abstract

Purpose : In many cell types, receptor-interacting protein kinase 3 (RIPK3) phosphorylates mixed lineage kinase domain-like protein (MLKL) to promote the necroptosis death pathway. However, RIPK3 has additional targets and non-necroptotic roles, particularly in endothelial cells (ECs). For example, our previous work shows that RIPK3 promotes developmental angiogenesis, including in the newborn mouse retina (PMID: 33449298). The current work studies the effect of endothelial RIPK3 on pathological angiogenesis associated with retinopathy of prematurity (ROP).

Methods : Endothelial cell-specific Ripk3 overexpression (Ripk3^oe/+;Cdh5(PAC)-Cre^ERT2 = Ripk3^iECoe), Ripk3 knockout (Ripk3^fl/fl;Cdh5(PAC)-Cre^ERT2 = Ripk3^iECko), and Mlkl overexpression (Mlkl^oe/+;Cdh5(PAC)-Cre^ERT2 = Mlkl^iECoe) mice were used in the oxygen-induced retinopathy (OIR) model. Cre-negative littermates were used as controls, and tamoxifen was administered via oral gavage daily (P10-12) in all mice. Neovascularization (NV) at P17 was quantified using SWIFT_NV plugin. Proliferation was measured by Ki67 immunostaining, and cell death was measured by cleaved caspase-3 immunostaining. In vitro, human WT RIPK3 and RIPK3-D160N (kinase dead) overexpression constructs were used for mechanistic studies in human primary retinal microvascular ECs.

Results : In the OIR model, we observed a marked reduction of NV area in Ripk3^iECoe mice compared to littermate controls. Comparable effects were not detected in Ripk3^iECko mice. There was also a significant decrease in proliferating ECs in Ripk3^iECoe retinas compared to littermate controls but no significant difference in apoptotic ECs. This suggests that RIPK3 overexpression suppresses endothelial proliferation associated with retinal ischemia. In addition, we observed a non-significant change of NV area in Mlkl^iECoe mice compared to littermate controls, indicating that RIPK3 suppresses pathological angiogenesis in a non-necroptotic manner.

Conclusions : Overexpression of endothelial RIPK3 suppresses pathological angiogenesis in a non-necroptotic manner. Future work studying the mechanism underlying the protective role of RIPK3 could pave the way for a novel therapeutic approach to prevent ROP.

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