Understanding the role of RIPK1 kinase activity in atherogenesis: a genetic versus pharmacological approach
Authors: Puylaert P , Coornaert I , Neutel CHG , Bertrand MJM , Guns PJ , De Meyer GRY , Martinet W 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): Fonds voor Wetenschappelijk Onderzoek (FWO)

Introduction
Necroptosis is a form of regulated necrosis mediated by the kinase activity of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and executed by RIPK3 and mixed lineage kinase domain like pseudokinase (MLKL). Because necroptosis is involved in necrotic core development in atherosclerotic plaques, we investigated the effect of a RIPK1 S25D/S25D mutation, which prevents the activation of RIPK1 kinase, on atherogenesis in ApoE-/- mice. Furthermore, we pharmacologically inhibited RIPK1 kinase activity by administering GSK’547 to ApoE-/- Fbn1 C1039G+/- mice, a mouse model of advanced atherosclerosis.

Purpose
We aimed to study the effect of RIPK1 kinase inhibition on atherogenesis through a genetic and a pharmacological approach in ApoE-/- and ApoE-/- Fbn1 C1039G+/- mice, respectively.

Methods
ApoE-/- and ApoE-/- Fbn1 C1039G+/- mice were fed a Western-type diet (WD) for up to 24 weeks and plaque necroptosis was confirmed. ApoE-/- Ripk1+/+ (n=16) and ApoE-/- Ripk1 S25D/S25D (n=12) mice were fed a WD for 16 weeks to induce plaque formation. ApoE-/- Fbn1 C1039G+/- mice received WD supplemented with GSK’547 (10 mg/kg BW/day, n=12-13/group) for 20 weeks to evaluate the effect of pharmacological RIPK1 kinase inhibition on atherogenesis.

Results
After 16 weeks WD, atherosclerotic plaques of ApoE-/- Ripk1 S25D/S25D mice were significantly larger as compared to ApoE-/- Ripk1+/+ mice (167±34 vs. 69±18 103 µm2, P=0.01). Absolute cell numbers (350±34 vs. 154±33 nuclei) and deposition of glycosaminoglycans (Alcian blue: 31±6 vs. 14±4%, P=0.023) were increased in plaques from ApoE-/- Ripk1 S25D/S25D mice while macrophage content (Mac3: 2.3±0.4 vs. 9.8±2.4%, P=0.012) was significantly decreased. Increased deposition of glycosaminoglycans was also observed in the vessel wall of ApoE-/- Ripk1 S25D/S25D mice (Alcian blue: 4.3±1.5 vs. 1.4±0.4 %, P=0.04). Together with increased internal elastic lamina area and increased vessel wall thickness, this points towards hypertrophic positive vascular remodeling in ApoE-/- Ripk1 S25D/S25D mice. Plaque apoptosis was not different between both groups. In contrast, pharmacological inhibition of RIPK1 kinase with GSK’547 in ApoE-/- Fbn1 C1039G+/- mice did not significantly alter plaque size and vessel properties after 20 weeks WD, but induced plaque apoptosis (TUNEL: 124±17 vs. 68±8 cells/mm2, P=0.018; cleaved caspase 3: 0.49±0.13 vs. 0.14±0.04 %; p=0.021).

Conclusion
Inhibition of RIPK1 kinase activity in ApoE-/- Ripk1 S25D/S25D mice accelerated plaque progression and induced positive vascular remodeling. These effects were not observed after pharmacological inhibition with GSK’547 in ApoE-/- Fbn1 C1039G+/- mice. However, a switch to apoptosis was observed after GSK’547 treatment, stressing the complex involvement of RIPK1 in cell survival and death.