Rationale: Cholesterol crystal embolism (CCE) can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown. Objective: We aimed to develop a new animal model of CCE to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure.Methods and Results: C57BL/6J mice were injected with CC into the left kidney artery. Primary endpoint was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, i.e. acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain-like-deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA (ecDNA). Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was < 3 h after CC injection. However, combining necrostatin-1s prophylaxis given 1 h before and DNase I 3 h after CC injection completely prevented kidney failure and infarcts. In vitro, CC did not directly induce plasmatic coagulation but induced neutrophil extracellular trap formation, and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. CC induced ATP release from aggregating platelets, which increased fibrin formation in a DNase-dependent manner. Conclusions: CC embolism causes arterial obstructions and organ failure via the formation of crytal clots with fibrin, platelets, and ecDNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.

中文翻译：

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晶体凝块作为胆固醇晶体栓塞的治疗靶标。

理由：胆固醇晶体栓塞（CCE）可能是威胁生命的晚期动脉粥样硬化并发症。病理生理学和治疗的分子靶标很大程度上未知。目的：我们旨在建立一种新的CCE动物模型，以剖析胆固醇晶体（CC）驱动的动脉闭塞，组织梗塞和器官衰竭的分子机制。方法和结果：向C57BL / 6J小鼠左侧注射CC肾动脉。主要终点是肾小球滤过率（GFR）。CC引起阻塞肾内动脉的晶体凝块和GFR的剂量依赖性下降，随后4周内GFR恢复，即急性肾脏疾病。相反，肾梗塞的程度变化更大。使用混合谱系激酶结构域类似缺陷型小鼠或necrostatin-1s治疗来阻断坏死病，可防止肾脏梗塞，但由于动脉阻塞持续而不受GFR的损失，晶体凝块是预防器官衰竭的主要靶标。CC涉及血小板，嗜中性粒细胞，纤维蛋白和细胞外DNA（ecDNA）。中性粒细胞耗竭或抑制中性粒细胞胞外释放的释放几乎没有影响，但是氯吡格雷对血小板P2Y12受体的拮抗作用，尿激酶的血纤蛋白溶解或重组DNase I的DNA消化均可以预防动脉阻塞，GFR丢失和肾梗塞。CC注射后机会窗口小于3小时。但是，将CC注射前1小时和DNase I 3小时后给予的necrostatin-1s预防结合起来，完全可以预防肾衰竭和梗塞。在体外，CC不直接诱导血浆凝结，但诱导中性粒细胞胞外陷阱形成，DNA主要从肾内皮细胞，中性粒细胞释放，而很少从血小板释放。CC诱导聚集血小板释放ATP，从而以DNase依赖性方式增加血纤蛋白的形成。结论：CC栓塞通过以纤维蛋白，血小板和ecDNA为关键成分的结晶凝块的形成引起动脉阻塞和器官衰竭。因此，我们的模型能够揭示CC栓塞综合征的发病机理，作为预防和靶向治疗的基础。CC诱导聚集血小板释放ATP，从而以DNase依赖性方式增加血纤蛋白的形成。结论：CC栓塞通过以纤维蛋白，血小板和ecDNA为关键成分的结晶凝块的形成引起动脉阻塞和器官衰竭。因此，我们的模型能够揭示CC栓塞综合征的发病机理，作为预防和靶向治疗的基础。CC诱导血小板聚集中的ATP释放，从而以DNase依赖性方式增加血纤蛋白的形成。结论：CC栓塞通过以纤维蛋白，血小板和ecDNA为关键成分的结晶凝块的形成引起动脉阻塞和器官衰竭。因此，我们的模型能够揭示CC栓塞综合征的发病机理，作为预防和靶向治疗的基础。