Abstract：
BACKGROUND: Macrophage-mediated cleaning up of dead cells is a crucial determinant in reducing coronary artery inflammation and maintaining vascular homeostasis. However, this process also leads to programmed death of macrophages. So far, the role of macrophage death in the progression of atherosclerosis remains controversial. Also, the underlying mechanism by which transcriptional regulation and reprogramming triggered by macrophage death pathways lead to changes in vascular inflammation and remodeling are still largely unknown. TRIM25-mediated RIG-I signaling plays a key role in regulation of macrophages fate, however the role of TRIM25 in macrophage death-mediated atherosclerotic progression remains unclear. This study aims to investigate the relationship between TRIM25 and macrophage death in atherosclerosis.
METHODS: A total of 34 blood samples of patients with coronary stent implantation, including chronic total occlusion (CTO) leisions (n = 14) or with more than 50% stenosis of a coronary artery but without CTO leisions (n = 20), were collected, and the serum level of TRIM25 was detected by ELISA. Apoe-/- mice with or without TRIM25 gene deletion were fed with the high-fat diet (HFD) for 12 weeks and the plaque areas, necrotic core size, aortic fibrosis and inflammation were investigated. TRIM25 wild-type and deficient macrophages were isolated, cultured and stimulated with ox-LDL, RNA-seq, real-time PCR, western blot and FACS experiments were used to screen and validate signaling pathways caused by TRIM25 deletion.
RESULTS: Downregulation of TRIM25 was observed in circulating blood of CTO patients and also in HFD-induced mouse aortas. After HFD for 12 weeks, TRIM25-/-ApoeE-/- mice developed smaller atherosclerotic plaques, less inflammation, lower collagen content and aortic fibrosis compared with TRIM25+/+ApoeE-/- mice. By RNA-seq and KEGG enrichment analysis, we revealed that deletion of TRIM25 mainly affected pyroptosis and necroptosis pathways in ox-LDL-induced macrophages, and the expressions of PARP1 and RIPK3, were significantly decreased in TRIM25 deficient macrophages. Overexpression of TRIM25 promoted M1 polarization and necroptosis of macrophages, while inhibition of PARP1 reversed this process. Further, we observed that XRCC1, a repairer of DNA damage, was significantly upregulated in TRIM25 deficient macrophages, inhibiting PARP1 activity and PARP1-mediated pro-inflammatory change, M1 polarization and necroptosis of macrophages. By contrast, TRIM25 overexpression mediated ubiquitination of XRCC1, and the inhibition of XRCC1 released PARP1, and activated macrophage M1 polarization and necroptosis, which accelerated aortic inflammation and atherosclerotic plaque progression.
CONCLUSIONS: Our study has uncovered a crucial role of the TRIM25-XRCC1Ub-PARP1-RIPK3 axis in regulating macrophage death during atherosclerosis, and we highlight the potential therapeutic significance of macrophage reprogramming regulation in preventing the development of atherosclerosis.
METHODS: A total of 34 blood samples of patients with coronary stent implantation, including chronic total occlusion (CTO) leisions (n = 14) or with more than 50% stenosis of a coronary artery but without CTO leisions (n = 20), were collected, and the serum level of TRIM25 was detected by ELISA. Apoe-/- mice with or without TRIM25 gene deletion were fed with the high-fat diet (HFD) for 12 weeks and the plaque areas, necrotic core size, aortic fibrosis and inflammation were investigated. TRIM25 wild-type and deficient macrophages were isolated, cultured and stimulated with ox-LDL, RNA-seq, real-time PCR, western blot and FACS experiments were used to screen and validate signaling pathways caused by TRIM25 deletion.
RESULTS: Downregulation of TRIM25 was observed in circulating blood of CTO patients and also in HFD-induced mouse aortas. After HFD for 12 weeks, TRIM25-/-ApoeE-/- mice developed smaller atherosclerotic plaques, less inflammation, lower collagen content and aortic fibrosis compared with TRIM25+/+ApoeE-/- mice. By RNA-seq and KEGG enrichment analysis, we revealed that deletion of TRIM25 mainly affected pyroptosis and necroptosis pathways in ox-LDL-induced macrophages, and the expressions of PARP1 and RIPK3, were significantly decreased in TRIM25 deficient macrophages. Overexpression of TRIM25 promoted M1 polarization and necroptosis of macrophages, while inhibition of PARP1 reversed this process. Further, we observed that XRCC1, a repairer of DNA damage, was significantly upregulated in TRIM25 deficient macrophages, inhibiting PARP1 activity and PARP1-mediated pro-inflammatory change, M1 polarization and necroptosis of macrophages. By contrast, TRIM25 overexpression mediated ubiquitination of XRCC1, and the inhibition of XRCC1 released PARP1, and activated macrophage M1 polarization and necroptosis, which accelerated aortic inflammation and atherosclerotic plaque progression.
CONCLUSIONS: Our study has uncovered a crucial role of the TRIM25-XRCC1Ub-PARP1-RIPK3 axis in regulating macrophage death during atherosclerosis, and we highlight the potential therapeutic significance of macrophage reprogramming regulation in preventing the development of atherosclerosis.
摘要：
背景：巨噬细胞介导的死细胞清理是减少冠状动脉炎症和维持血管稳态的关键决定因素。然而,这个过程也会导致巨噬细胞的程序性死亡。到目前为止,巨噬细胞死亡在动脉粥样硬化进展中的作用仍存在争议.此外，由巨噬细胞死亡途径触发的转录调控和重编程导致血管炎症和重塑改变的潜在机制仍在很大程度上未知.TRIM25介导的RIG-I信号在调节巨噬细胞命运中起关键作用，然而,TRIM25在巨噬细胞死亡介导的动脉粥样硬化进展中的作用尚不清楚.本研究旨在探讨动脉粥样硬化中TRIM25与巨噬细胞死亡的关系。
方法：共34例冠状动脉支架植入术患者的血液样本，包括慢性完全闭塞（CTO）病变（n=14）或冠状动脉狭窄超过50％但无CTO病变（n=20），被收集,ELISA法检测血清TRIM25水平。用高脂饮食(HFD)喂养有或没有TRIM25基因缺失的Apo-/-小鼠12周，坏死核大小，研究了主动脉纤维化和炎症。分离TRIM25野生型和缺陷型巨噬细胞，用ox-LDL培养和刺激，RNA-seq,实时PCR，采用westernblot和FACS实验筛选和验证TRIM25缺失引起的信号通路.
结果：在CTO患者的循环血液中以及在HFD诱导的小鼠主动脉中观察到TRIM25的下调。HFD12周后，TRIM25-/-ApoeE-/-小鼠出现较小的动脉粥样硬化斑块，更少的炎症，与TRIM25+/+ApoeE-/-小鼠相比，胶原含量和主动脉纤维化较低。通过RNA-seq和KEGG富集分析，我们发现，TRIM25的缺失主要影响ox-LDL诱导的巨噬细胞的焦凋亡和坏死途径，TRIM25缺陷型巨噬细胞中PARP1和RIPK3的表达显著降低。TRIM25过表达促进巨噬细胞M1极化和坏死,而抑制PARP1逆转了这一过程。Further,我们观察到XRCC1，DNA损伤的修复者，在缺乏TRIM25的巨噬细胞中显著上调，抑制PARP1活性和PARP1介导的促炎变化，巨噬细胞的M1极化和坏死。相比之下,TRIM25过表达介导XRCC1的泛素化，抑制XRCC1释放PARP1，激活巨噬细胞M1极化和坏死，加速主动脉炎症和动脉粥样硬化斑块进展。
结论：我们的研究揭示了TRIM25-XRCC1Ub-PARP1-RIPK3轴在动脉粥样硬化过程中调节巨噬细胞死亡的关键作用，我们强调了巨噬细胞重编程调节在预防动脉粥样硬化发展中的潜在治疗意义。
方法：共34例冠状动脉支架植入术患者的血液样本，包括慢性完全闭塞（CTO）病变（n=14）或冠状动脉狭窄超过50％但无CTO病变（n=20），被收集,ELISA法检测血清TRIM25水平。用高脂饮食(HFD)喂养有或没有TRIM25基因缺失的Apo-/-小鼠12周，坏死核大小，研究了主动脉纤维化和炎症。分离TRIM25野生型和缺陷型巨噬细胞，用ox-LDL培养和刺激，RNA-seq,实时PCR，采用westernblot和FACS实验筛选和验证TRIM25缺失引起的信号通路.
结果：在CTO患者的循环血液中以及在HFD诱导的小鼠主动脉中观察到TRIM25的下调。HFD12周后，TRIM25-/-ApoeE-/-小鼠出现较小的动脉粥样硬化斑块，更少的炎症，与TRIM25+/+ApoeE-/-小鼠相比，胶原含量和主动脉纤维化较低。通过RNA-seq和KEGG富集分析，我们发现，TRIM25的缺失主要影响ox-LDL诱导的巨噬细胞的焦凋亡和坏死途径，TRIM25缺陷型巨噬细胞中PARP1和RIPK3的表达显著降低。TRIM25过表达促进巨噬细胞M1极化和坏死,而抑制PARP1逆转了这一过程。Further,我们观察到XRCC1，DNA损伤的修复者，在缺乏TRIM25的巨噬细胞中显著上调，抑制PARP1活性和PARP1介导的促炎变化，巨噬细胞的M1极化和坏死。相比之下,TRIM25过表达介导XRCC1的泛素化，抑制XRCC1释放PARP1，激活巨噬细胞M1极化和坏死，加速主动脉炎症和动脉粥样硬化斑块进展。
结论：我们的研究揭示了TRIM25-XRCC1Ub-PARP1-RIPK3轴在动脉粥样硬化过程中调节巨噬细胞死亡的关键作用，我们强调了巨噬细胞重编程调节在预防动脉粥样硬化发展中的潜在治疗意义。
