RIPK1 Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice

Circulation. 2021 Jan 12;143(2):163-177. doi: 10.1161/CIRCULATIONAHA.118.038379. Epub 2020 Nov 23.

Abstract

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis.

Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe-/- mice fed a cholesterol-rich (Western) diet for 8 weeks.

Results: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin, and monocyte attachment.

Conclusions: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Keywords: NF-kappa B; RIPK1 protein, human; atherosclerosis; inflammation; macrophages.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Atherosclerosis / genetics
  • Atherosclerosis / metabolism*
  • Atherosclerosis / pathology
  • Cells, Cultured
  • Cholesterol, Dietary / administration & dosage
  • Cholesterol, Dietary / adverse effects
  • Female
  • Gene Expression
  • Gene Silencing / physiology*
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Inflammation / genetics
  • Inflammation / metabolism
  • Inflammation / pathology
  • Inflammation Mediators / antagonists & inhibitors
  • Inflammation Mediators / metabolism*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / genetics
  • NF-kappa B / metabolism*
  • Receptor-Interacting Protein Serine-Threonine Kinases / biosynthesis*
  • Receptor-Interacting Protein Serine-Threonine Kinases / genetics

Substances

  • Cholesterol, Dietary
  • Inflammation Mediators
  • NF-kappa B
  • RIPK1 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk1 protein, mouse