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Circ Res. 2020 Feb 24. doi: 10.1161/CIRCRESAHA.119.315625. [Epub ahead of print]

Crystal Clots as Therapeutic Target in Cholesterol Crystal Embolism.

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Klinikum der Universtät München, LMU.
Klinikum der Universität München, LMU.
Pharmacology Division, CSIR Central Drug Research Institute, INDIA.
Institute for Pathology, RWTH Aachen University Hospital, GERMANY.
Radiology, University Hospital Erlangen, GERMANY.
Excellence Centre for Research, University of Florence, ITALY.
Institute for Pathology and Department of Nephrology, RWTH University, GERMANY.
nIstitute for Pathology and Department of Nephrology, RWTH University, GERMANY.
University of Würzburg, Rudolf Virchow Center.
Medizinische Klinik und Poliklinik IV, University of Munich, GERMANY.
Biochemistry, Maastricht University, NETHERLANDS.
Biochemistry, Cardiovascular Research Institute Maastricht, NETHERLANDS.
Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig-Maximilians University Munich.
Experimental Molecular Imaging, RWTH Aachen, GERMANY.
Pathology and Nephrology, University Hospital of the RWTH Aachen, GERMANY.
Klinikum der Universität München, LMU.


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.


Acute kidney injury; Atheroembolism; Necroptosis; Platelets

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