From the Medizinische Klinik und Poliklinik IV, Klinikum der Universität, LMU München, Germany (C.S., T.K., S.S., S.R.M., L.Y., H.-J.A.).
Department of Nephrology, Institute of Pathology (B.M.K, P.B.), RWTH Aachen University Hospital, Germany.
Circ Res. 2020 Apr 10;126(8):e37-e52. doi: 10.1161/CIRCRESAHA.119.315625. Epub 2020 Feb 24.
Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown.
We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure.
C57BL/6J mice were injected with CC into the left kidney artery. Primary end point 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, that is, 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. 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 hours after CC injection. However, combining Nec-1s (necrostatin-1s) prophylaxis given 1 hour before and DNase I 3 hours 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.
CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA 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.
胆固醇结晶栓塞是晚期动脉粥样硬化的一种危及生命的并发症。其病理生理学和治疗的分子靶点在很大程度上尚不清楚。
我们旨在开发一种新的胆固醇结晶栓塞动物模型，以剖析胆固醇结晶（CC）驱动的动脉闭塞、组织梗死和器官衰竭的分子机制。
C57BL/6J 小鼠被注射 CC 到左肾动脉。主要终点是肾小球滤过率（GFR）。CC 导致结晶栓子阻塞肾内动脉，并导致 GFR 呈剂量依赖性下降，随后在 4 周内恢复 GFR，即急性肾损伤。相比之下，肾脏梗死的程度变化较大。用混合谱系激酶结构域样缺失小鼠或 Necrostatin-1s 治疗阻断坏死性凋亡可防止肾梗死，但不能防止 GFR 丧失，因为动脉阻塞仍然存在，这表明结晶栓子是防止器官衰竭的主要靶标。CC 涉及血小板、中性粒细胞、纤维蛋白和细胞外 DNA。中性粒细胞耗竭或抑制中性粒细胞胞外诱捕网的释放几乎没有效果，但用氯吡格雷拮抗血小板 P2Y12 受体、用尿激酶溶解纤维蛋白或用重组 DNA 酶 I 消化 DNA 均可防止动脉阻塞、GFR 丧失和肾脏梗死。CC 注射后<3 小时是治疗的机会窗口。然而，在 CC 注射前 1 小时给予 Nec-1s（Necrostatin-1s）预防，并在 CC 注射后 3 小时给予 DNA 酶 I，可完全预防肾衰和梗死。在体外，CC 不会直接诱导血浆凝固，但会诱导中性粒细胞胞外诱捕网的形成和主要来自肾脏内皮细胞、中性粒细胞和少数血小板的 DNA 释放。CC 从聚集的血小板中诱导 ATP 释放，以依赖于 DNA 酶的方式增加纤维蛋白的形成。
CC 栓塞通过形成含有纤维蛋白、血小板和细胞外 DNA 的结晶栓子导致动脉阻塞和器官衰竭，这些是关键成分。因此，我们的模型可以揭示 CC 栓塞综合征的发病机制，为预防和靶向治疗提供基础。
