University of Ottawa Heart Institute, Canada (D.K., M.-A.N., M.G., Z.L., H.W., J.W.K., R.J., A.M., A.R., P.L., K.J.R.).
Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia (D.K., N.O., P.E., E.G.).
Circulation. 2021 Jan 12;143(2):163-177. doi: 10.1161/CIRCULATIONAHA.118.038379. Epub 2020 Nov 23.
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.
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 mice fed a cholesterol-rich (Western) diet for 8 weeks.
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, <0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], <0.05) in comparison with controls. 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 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of , , and monocyte attachment.
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.
固有免疫系统的慢性激活会引发炎症，并直接导致动脉粥样硬化。我们之前曾表明，在致动脉粥样硬化斑块中，巨噬细胞会对氧化型低密度脂蛋白和损伤相关分子模式等无菌配体产生 RIPK3（受体相互作用丝氨酸/苏氨酸蛋白激酶 3）-MLKL（混合谱系激酶结构域样蛋白）依赖性程序性坏死，并且坏死在晚期动脉粥样硬化斑块中是活跃的。RIPK3-MLKL 坏死机制的上游是 RIPK1（受体相互作用丝氨酸/苏氨酸蛋白激酶 1），它作为一个主开关，控制细胞在受到细胞外刺激时是通过 NF-κB（核因子 κ-轻链增强子的 B 细胞激活）依赖性炎症、半胱天冬酶依赖性细胞凋亡还是坏死来反应。因此，我们着手研究 RIPK1 在动脉粥样硬化发展中的作用，该疾病在早期主要由 NF-κB 依赖性炎症驱动。我们假设，与 RIPK3 和 MLKL 不同，RIPK1 主要在早期致动脉粥样硬化病变中驱动 NF-κB 依赖性炎症，而敲低 RIPK1 将减少炎症细胞的激活并防止动脉粥样硬化的进展。
我们检测了人及鼠动脉粥样硬化病变中 RIPK1 蛋白和 mRNA 的表达，并在体外通过巨噬细胞和内皮细胞的功能丧失方法来测量炎症反应。我们给喂饲富含胆固醇（西方）饮食 8 周的小鼠每周注射 RIPK1 反义寡核苷酸。
我们发现 RIPK1 在人及鼠的早期动脉粥样硬化病变中表达丰富。与对照组相比，RIPK1 反义寡核苷酸的治疗导致主动脉窦和动脉粥样硬化病变面积减少（分别减少 47.2%或 58.8%，<0.01），以及血浆炎症细胞因子（白细胞介素 1α[IL-1α]、白细胞介素 17A[IL-17A]，<0.05）。巨噬细胞中的 敲低减少了炎症基因（NF-κB、肿瘤坏死因子 α[TNFα]、白细胞介素 1α），并降低了体内脂多糖和致动脉粥样硬化饮食诱导的 NF-κB 激活。在内皮细胞中，TNFα 诱导的 NF-κB 向核内易位时， 敲低阻止了 NF-κB 的易位，相应地减少了 、 和单核细胞附着的基因表达。
我们通过其激活 NF-κB 途径和促进炎症细胞因子释放的能力，将 RIPK1 确定为动脉粥样硬化中炎症的核心驱动因素。鉴于人动脉粥样硬化病变中 RIPK1 的高表达，我们的研究表明 RIPK1 可作为降低高危冠心病患者残余炎症的未来治疗靶点。
