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

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-kappa B (nuclear factor kappa-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-kappa B-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-kappa B-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-1 alpha [interleukin 1 alpha], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-kappa B, TNF alpha [tumor necrosis factor alpha], IL-1 alpha) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-kappa B activation. In endothelial cells, knockdown of RIPK1 prevented NF-kappa B translocation to the nucleus in response to TNF alpha, 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-kappa B 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.

Automated summary

The study indicates that RIPK1 plays a central role in driving inflammation in atherosclerosis by activating the NF-kappa B pathway and promoting the release of inflammatory cytokines. Knockdown of RIPK1 reduces inflammatory cell activation and protects against the progression of atherosclerosis. These findings suggest RIPK1 as a potential therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.