Extracellular DNA contributes to cholesterol crystal embolism-induced clot formation, acute kidney injury, and tissue infarction

Atherosklerose ist weltweit eine der Hauptursachen für Morbidität und Mortalität. Bei fortgeschrittener Atherosklerose ist die Cholesterinkristallembolie (CCE) eine potenziell lebensbedrohliche Komplikation mit einer durchschnittlichen Mortalität von 62,8%. Autopsien oder Gewebebiopsien zeigen Cholesterinkristalle (CC) im arteriellen Lumen, umgeben von einer fibrotischen Matrix, die das Gefäßlumen verschließt. Über die genauen zellulären und molekularen Mechanismen nach CCE ist wenig bekannt, was teilweise auf das Fehlen eines Tiermodells zurückzuführen ist. Wir stellten daher die Hypothese auf, dass die Entwicklung eines reproduzierbaren Mausmodells der CCE zur Nachahmung der morphologischen und funktionellen Eigenschaften der CCE beim Menschen dazu beitragen würde, die molekularen Mechanismen des CC-gesteuerten arteriellen Verschlusses, des Gewebeinfarkts und des Organversagens zu untersuchen. CCE wurden in C57BL/6J-Mäusen durch Injektion von CC über einen minimal- invasiven Eingriff in die linke Nierenarterie induziert. Primärer Endpunkt war die glomeruläre Filtrationsrate (GFR), die am wachen und frei beweglichen Tier gemessen wurde, um den Abfall der exkretorischen Nierenfunktion als Marker eines akuten Nierenversagens zu bestimmten. Die Größe des Niereninfarkts wurde, wie bei Myokardinfarkt oder Schlaganfallmodellen etabliert, per TTC-Färbung von Nierenschnitten und Planimetrie quantifiziert. Injektion von CC verursachte einen dosis-abhängigen Abfall der GFR und Territorialinfarkte der Niere. Ursache waren Verschlüsse präglomerulärer Arterien und Arteriolen. Der Kristallanteil am Gefäßverschluss war gering, stattdessen fanden sich Fibrin+ Thrombusmaterial um die Kristalle, die das arterielle Lumen ausfüllten. Wir nannten diese Strukturen “Kristallthrombosen”. Im Vergleich zum GFR Abfall, war das Ausmaß der Infarktgrößen variabler. 3D Rekonstruktionen von Angio-μCTs zeigte partielle und vollständige arterielle Verschlüsse und Rarefizierung der arteriellen Blutgefäße. Histologisch fand sich nach 24 h ein deutliches perilesionales Neutrophileninfiltrat. Somit imitiert unser Modell periphere CCE mit arteriellen Verschlüssen, die akute territoriale Infarkte, perilesionale Entzündung und fiunktionelles Organversagen. Das Blockieren der Nekroinflammation/Infarzierung in mixed lineage kinase domain-like (Mlkl)-defizienten Mäusen oder mit dem Inhibitor Nec-1s bzw. einem NLRP3-Inhibitor reduzierte signifikant die Infarktgröße und Infiltration von Neutrophilen im Vergleich zu Kontrollen. Keine dieser Interventionen hatte jedoch Auswirkungen auf den durch CCE verursachten GFR-Verlust (=Organversagen), weil keine der Interventionen Einfluss auf die arteriellen Verschlüsse hatte. Da in der Niere die Funktion zu allererst von der glomerulären Perfusion abhängt, verhindert die Hemmung der ischämen Nekrose alleine noch nicht das Nierenversagen. Somit war klar, dass die Kristallthrombosen das entscheidende Therapietarget bei CCE darstellen. Histologisch bestanden die Kristallthrombosen aus Erythrozyten, Plättchen, Neutrophile, Fibrin, und extrazelluläre DNA (ecDNA). Zunächst haben wir Neutrophile mit einem depletierenden Antikörper selektiv entfernt, bzw. die Bildung von neutrophil extracellular traps (NETs) mit einem Inhibitor gehemmt. Beide Interventionen verringerten die Größe des Niereninfarkts im Vergleich zur Kontrollgruppe, dies hatte jedoch keinen signifikanten Einfluss auf die arteriellen Verschlüsse bzw. den GFR-Verlust. Im Gegensatz dazu schützte der Thrombozyten-P2Y12-Rezeptorantagonist Clopidogrel Mäuse vollständig vor Kristallthrombosen, GFR-Abfall und Niereninfarkt. Daher sind Blutplättchen, jedoch nicht neutrophile Granulozyten, von zentraler Bedeutung für CCE-induzierte Kristallthrombosen und ihre Folgen. Als Nächstes testeten wir die Wirkung von Heparin und des Fibrinolytikums Urokinase. Nach 24 Stunden reduzierten sowohl Heparin als auch Urokinase die Anzahl der arteriellen Verschlüsse signifikant. Niereninfarkt, Nierenverletzung, Infiltration von Neutrophilen, Gefäßverletzung sowie tubuläre Nekrose waren nahezu vollständig abwesend. Beide Behandlungen hatten im Vergleich zu mit Vehikel-behandelten Mäusen einen vollständigen Schutz vor GFR-Abfall. Zusammenfassend lässt sich sagen, dass nicht die Kristalle an sich, sondern die Kristallthrombosen arterielle Obstruktion, Gewebeinfarkt und Organversagen verursachen. Um die Bedeutung der ecDNA zu untersuchen, gaben wir rekombinante DNase I, die ecDNA degradiert. Tatsächlich war 24 h nach DNase I Gabe in den Arterien keine ecDNA mehr nachweisbar und, überraschenderweise, traten auch Kristallthrombosen nicht mehr auf. Die Verhinderung von arteriellen Verschlüssen war mit einem vollständigen Schutz vor GFR-Abfall, einer signifikanten Verringerung der Niereninfarktgröße verbunden. Somit ist ecDNA eine weitere nicht-redundante Komponente der CCE-bedingten arteriellen Obstruktion, des Gewebeinfarkts und des Organversagens. Da Herz- oder Aortenoperationen die Verwendung von Antikoagulanzien oder Fibrinolytika ausschließen, betrachteten wir rekombinante DNase I als mögliche Alternative zur Abschwächung der CC-Gerinnselbildung durch Hemmung der Fibrinbildung und der ecDNA-Akkumulation. Zuerst testeten wir das therapeutische Zeitfenster und stellten fest, dass die DNase I-Behandlung, die 3 Stunden nach der CCE verabreicht wurde, immer noch einen Trend zu verbesserten Ergebnissen im Vergleich zu 6 und 12 Stunden zeigte. Um die Ergebnisse bei der Einstellung einer CCE im Zusammenhang mit kardiovaskulären Eingriffen weiter zu optimieren, haben wir ein Regime getestet, das eine präventive Einzeldosis des Nekroptosehemmers Nec-1s mit therapeutischer Gabe von rekombinanter DNase 1 3 Stunden nach Kristallinjektion kombiniert. Hierunter kam es zu einer vollständigen Protektion von Kristallthrombosen Organversagen und Infarkt, was eine neue Behandlungsoption bei elektiven Eingriffen bei Hochrisikopatienten aufzeigen könnte. Mechanistische in vitro Untersuchungen im Organ-Chip Modell, zeigten wie Cholesterin-Kristalle zu Endothelschäden führen, dass ecDNA v.a. aus Endothel und Neutrophilen freigesetzt wird. Thrombozyten setzen nur wenig mitochondriale DNA frei. DNase I inhibiert die CC-induzierte Thrombozytenaktivierung, möglicherweise durch Abbau von ADP. Zusammengenommen präsentieren wir erstmals ein Mausmodell einer CCE mit Organversagen und Infarkt, das pathophysiologische Studien gestattet. Nicht der Infarkt an sich, sondern die Kristallthrombosen sind für das Organversagen entscheidend. Endothelschädigung mit Freisetzung von ecDNA und Plättchenaktivierung führen zur Bildung der Kristallthrombosen und bieten alte und neue Therapietargets. Eine prophylaktische Einmalgabe eines Zelltodinhibitors und die Gabe von DNase I im postinterventionellen Zeitfenster von 3 h (bei der Maus) könnten helfen, die Prognose von Patienten mit Prozedur-assoziierter CCE zu verbessern.Atherosclerosis is a leading cause of global morbidity and mortality. In advanced atherosclerosis, cholesterol crystal (CC) embolism (CCE) is a potentially life-threatening complication with an average mortality of 62.8 %. Autopsies or tissue biopsies reveal CC inside the arterial lumen surrounded by an undefined biological matrix obstructing the vessel lumen. Little is known about the precise cellular and molecular mechanisms following CCE, in part due to the lack of animal models. Therefore, I hypothesized that developing a reproducible mouse model of CCE to mimic the morphological and functional characteristics of CCE in humans would be instrumental to dissect the molecular mechanisms of CC-driven arterial occlusion, tissue infarction, and organ failure. To induce CCE, different doses of CC were injected into the left kidney artery of C57BL/6J mice. Acute kidney failure was evaluated by kidney function (i.e. GFR), kidney infarction was quantified using the TTC method. CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR. In contrast, the extent of kidney infarction was more variable. 3D μCT showed partial and complete arterial occlusions, blood vessel rarefaction, and volume change. The macroscopic analysis revealed kidney swelling and territorial infarctions, tubular necrosis, interstitial edema, neutrophil infiltrates, and loss of CD31. Thus, intraarterial CC injection induces arterial occlusions causing acute territorial infarctions, perilesional inflammation, and organ failure. Blocking necroptosis with Mlkl-/- mice or Nec-1s, and the NLRP3 inhibitor significantly reduced infarct size, kidney injury, and neutrophil infiltration at 24 h compared to WT controls. However, none of these interventions affected CCE-related GFR loss. Consistently, necroinflammation is involved in kidney infarction but not in arterial occlusions as an upstream event. Thus, as nephron perfusion is ultimately required for kidney function, inhibiting infarction alone does not prevent acute kidney failure. Immunostaining revealed that crystal clots involved platelets, neutrophils, fibrin, and extracellular DNA (ecDNA). Therefore, I depleted neutrophils or inhibited NET formation before CC injection. Neutrophil depletion or NET inhibition significantly decreased kidney infarction compared to the control groups, but this had no significant effect on arterial obstructions or GFR loss, maybe because mononuclear cells had partially replaced neutrophils inside crystal clots as a source of ecDNA. In contrast, the platelet P2Y12 receptor antagonist clopidogrel completely protected mice from intravascular obstructions, GFR loss, kidney infarction, and perilesional neutrophil infiltrate. Therefore, CC occlude arteries by forming crystal clots consisting of fibrin, platelets, and neutrophils. Thus, platelets but not neutrophils are central for CCE-related arterial occlusion, organ failure, and tissue infarction. Next, I tested the effects of the anticoagulant heparin and the fibrinolytic agent urokinase. At 24h both heparin and urokinase significantly reduced the arterial occlusions number, kidney infarction, kidney injury, neutrophils infiltration, vascular injury as well as tubular necrosis. Both treatments had complete protection from GFR loss compared to vehicle-treated mice. Conclusively, not the crystals per se but rather crystal clots cause arterial obstruction, tissue infarction, and organ failure. In the DNase I treatment group, I noticed a significant reduction in the percentage of CC clots with ecDNA. After 24h of DNase I treatment intraarterial ecDNA had disappeared and the number of arterial occlusions was significantly reduced. Preventing arterial occlusions was associated with complete protection from GFR loss, a significant reduction in kidney infarct size as well as kidney cell death, neutrophil infiltrates, and vascular rarefaction. Thus, ecDNA is another non-redundant component of CCE-related arterial obstruction, tissue infarction, and organ failure. As cardiac or aorta surgeries preclude the use of anticoagulants or fibrinolytic agents, I considered recombinant DNase I as a possible alternative to attenuate CC clot formation by inhibiting fibrin formation and ecDNA accumulation. First, I tested the therapeutic window-of-opportunity and found that DNase I treatment given 3 h after CCE showed trends towards improved outcomes compared to 6 h and 12 h. To further optimize outcomes in the setting of a cardiovascular procedure-related CCE I tested a regimen combining a pre-emptive single dose of the necroptosis inhibitor Nec-1s with therapeutic recombinant DNase I gave 3 h after intraarterial CC injection. This approach could be feasible as prophylaxis given to all patients at risk, while DNase I would be only given to those with signs of CCE into the kidney, e.g. an early decline of urinary output. This dual strategy resulted in significant protection from GFR loss and kidney infarction in almost all animals together with a significant reduction in vascular occlusions by crystal clots. In my in vitro studies, platelets were exposed to thrombin with or without CC and found that CC enhances fibrinogen release from platelet alpha (α)-granules, which further promotes fibrin clot formation. CC exposure also induced ATP secretion from dense (δ)-granules, but co-incubation with DNase I strongly reduced these extracellular ATP releases. Next, I stimulated platelets with thrombin and collagen-related peptide and indicated DNase I can inhibit fibrinogen and ATP secretion and subsequent fibrin formation and P2Y12 receptor signaling, respectively. To model this process in vitro, I tested collagen-driven platelet aggregation with or without CC. Indeed, collagen I triggered massive platelet aggregation within 5 min with CC, DNase I treatment normalized this accelerated aggregation response. Endothelial cells and neutrophils studies showed that CC did not directly induce plasmatic coagulation but induced NET formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. Thus, the in vitro studies support that CC and platelet dependent ecDNA release from neutrophils and endothelial cells, and DNase I can attenuate CC-induced platelet activation, aggregation, and fibrin clot formation. In summary, not CC by itself but the fibrin clots forming around CC obstruct peripheral arteries causing tissue infarction and organ failure. Hence, crystal clots represent the primary target for therapeutic interventions. Among the possible molecular targets in thrombosis and haemostasis, especially enhancing fibrinolysis or inhibiting platelet purinergic signaling could reduce arterial occlusions, infarction, and organ failure albeit with a relatively short window-of-opportunity up to 3 h. My results suggest that prophylactic necroptosis inhibition with a combination of DNase I therapy could have a synergistic effect on CC induced clot formation in mice and might be a feasible two-step prophylactic/therapeutic approach in human patients with a risk for procedure-related CCE

A guide to crystal‐related and nano‐ or microparticle‐related tissue responses

Crystals and nano‐ and microparticles form inside the human body from intrinsic proteins, minerals, or metabolites or enter the body as particulate matter from occupational and environmental sources. Associated tissue injuries and diseases mostly develop from cellular responses to such crystal deposits and include inflammation, cell necrosis, granuloma formation, tissue fibrosis, and stone‐related obstruction of excretory organs. But how do crystals and nano‐ and microparticles trigger these biological processes? Which pathomechanisms are identical across different particle types, sizes, and shapes? In addition, which mechanisms are specific to the atomic or molecular structure of crystals or to specific sizes or shapes? Do specific cellular or molecular mechanisms qualify as target for therapeutic interventions? Here, we provide a guide to approach this diverse and multidisciplinary research domain. We give an overview about the clinical spectrum of crystallopathies, about shared and specific pathomechanisms as a conceptual overview before digging deeper into the specialty field of interest

Extracellular DNA-A Danger Signal Triggering Immunothrombosis

Clotting and inflammation are effective danger response patterns positively selected by evolution to limit fatal bleeding and pathogen invasion upon traumatic injuries. As a trade-off, thrombotic, and thromboembolic events complicate severe forms of infectious and non-infectious states of acute and chronic inflammation, i.e., immunothrombosis. Factors linked to thrombosis and inflammation include mediators released by platelet granules, complement, and lipid mediators and certain integrins. Extracellular deoxyribonucleic acid (DNA) was a previously unrecognized cellular component in the blood, which elicits profound proinflammatory and prothrombotic effects. Pathogens trigger the release of extracellular DNA together with other pathogen-associated molecular patterns. Dying cells in the inflamed or infected tissue release extracellular DNA together with other danger associated molecular pattern (DAMPs). Neutrophils release DNA by forming neutrophil extracellular traps (NETs) during infection, trauma or other forms of vascular injury. Fluorescence tissue imaging localized extracellular DNA to sites of injury and to intravascular thrombi. Functional studies using deoxyribonuclease (DNase)-deficient mouse strains or recombinant DNase show that extracellular DNA contributes to the process of immunothrombosis. Here, we review rodent models of immunothrombosis and the evolving evidence for extracellular DNA as a driver of immunothrombosis and discuss challenges and prospects for extracellular DNA as a potential therapeutic target

Neutrophil circadian rhythm is associated with different outcomes of acute kidney injury due to cholesterol crystal embolism

Cholesterol crystal (CC) embolism can cause acute tissue infarction and ischemic necrosis via triggering diffuse thrombotic angiopathy occluding arterioles and arteries. Neutrophils contribute to crystal-induced immunothrombosis as well as to ischemic necrosis-related necroinflammation. We speculated that CC embolism-induced acute kidney injury (AKI) would be circadian rhythm-dependent and associated with cyclic differences in neutrophil function. Injection of CC into the left kidney induced thrombotic angiopathy progressing starting as early as 3 h after CC injection followed by a progressive ischemic cortical necrosis and AKI at 24 h. In C57BL/6J mice, circulating CD11b+Ly6G+ neutrophils were higher during the day phase [Zeitgeber time (ZT) 0–12] compared to the dark phase (ZT12-24). In the time frame of thrombus formation at ZT13, more neutrophils were recruited into the injured kidney 24 h later compared to CC embolism at ZT5. This effect was associated with an increased circulating number of CXCR2+ neutrophils as well as an upregulated kidney adhesion molecule and chemokine expression. These findings were associated with a significant increase in kidney necrosis, and endothelial injury at ZT13. Thus, the time of day has an effect also on CC embolism-related AKI in association with the circadian rhythm of neutrophil recruitment

IRF8-Dependent Type I Conventional Dendritic Cells (cDC1s) Control Post-Ischemic Inflammation and Mildly Protect Against Post-Ischemic Acute Kidney Injury and Disease

Post-ischemic acute kidney injury and disease (AKI/AKD) involve acute tubular necrosis and irreversible nephron loss. Mononuclear phagocytes including conventional dendritic cells (cDCs) are present during different phases of injury and repair, but the functional contribution of this subset remains controversial. Transcription factor interferon regulatory factor 8 (IRF8) is required for the development of type I conventional dendritic cells (cDC1s) lineage and helps to define distinct cDC1 subsets. We identified one distinct subset among mononuclear phagocyte subsets according to the expression patterns of CD11b and CD11c in healthy kidney and lymphoid organs, of which IRF8 was significantly expressed in the CD11blowCD11chigh subset that mainly comprised cDC1s. Next, we applied a Irf8-deficient mouse line (Irf8fl/flClec9acre mice) to specifically target Clec9a-expressing cDC1s in vivo. During post-ischemic AKI/AKD, these mice lacked cDC1s in the kidney without affecting cDC2s. The absence of cDC1s mildly aggravated the loss of living primary tubule and decline of kidney function, which was associated with decreased anti-inflammatory Tregs-related immune responses, but increased T helper type 1 (TH1)-related and pro-inflammatory cytokines, infiltrating neutrophils and acute tubular cell death, while we also observed a reduced number of cytotoxic CD8+ T cells in the kidney when cDC1s were absent. Together, our data show that IRF8 is indispensable for kidney cDC1s. Kidney cDC1s mildly protect against post-ischemic AKI/AKD, probably via suppressing tissue inflammation and damage, which implies an immunoregulatory role for cDC1s

IL-22 sustains epithelial integrity in progressive kidney remodeling and fibrosis

IL-22, a member of the IL-10 cytokine family, accelerates tubule regeneration upon acute kidney injury, hence we speculated on a protective role also in chronic kidney disease. We quantified intrarenal IL-22 expression after unilateral ureteral (UUO) in wild-type mice and performed UUO in IL-22 knock-out animals. Obstruction phenotypic differences between IL22(+/+) and IL22(-/-) mice were assessed by histology, immunohistochemistry, immunofluorescence as well as western blotting and reverse-transcriptase quantitative PCR ex vivo. Additionally, we performed in vitro experiments using both murine and human tubular cells to characterize IL-22 effects in epithelial healing. We found increasing IL22 positivity in infiltrating immune cells over time upon UUO in wild-type mice. UUO in IL22(-/-) mice caused more tubular cell injury as defined by TUNEL positive cells and loss of tetragonolobus lectin staining. Instead, tubular dilation, loss of CD31+ perivascular capillaries, and interstitial fibrosis were independent of the Il22 genotype as assessed by standard histology, immunostaining, and mRNA expression profiling. In vitro experiments showed that recombinant human IL-22 significantly enhanced human tubular epithelial cell proliferation and wound closure upon mechanical injury, and electric cell-substrate impedance sensing studies revealed that recombinant IL-22 sustained tubular epithelial barrier function upon injury. In contrast, IL-22 had no such direct effects on human fibroblasts. Together, in progressive kidney remodeling upon UUO, infiltrating immune cells secrete IL-22, which augments tubular epithelial integrity and epithelial barrier function, but does not affect vascular rarefaction or fibrogenesis. We conclude that IL-22 could represent a molecular target to specifically modulate tubular atrophy

Mitochondria Permeability Transition versus Necroptosis in Oxalate-Induced AKI

Serum oxalate levels suddenly increase with certain dietary exposures or ethylene glycol poisoning and are a well known cause of AKI. Established contributors to oxalate crystal-induced renal necroinflammation include the NACHT, LRR and PYD domains-containing protein-3 (NLRP3) inflammasome and mixed lineage kinase domain-like (MLKL) protein-dependent tubule necroptosis. These studies examined the role of a novel form of necrosis triggered by altered mitochondrial function. METHODS: To better understand the molecular pathophysiology of oxalate-induced AIK, we conducted in vitro studies in mouse and human kidney cells and in vivo studies in mice, including wild-type mice and knockout mice deficient in peptidylprolyl isomerase F (Ppif) or deficient in both Ppif and Mlkl. RESULTS: Crystals of calcium oxalate, monosodium urate, or calcium pyrophosphate dihydrate, as well as silica microparticles, triggered cell necrosis involving PPIF-dependent mitochondrial permeability transition. This process involves crystal phagocytosis, lysosomal cathepsin leakage, and increased release of reactive oxygen species. Mice with acute oxalosis displayed calcium oxalate crystals inside distal tubular epithelial cells associated with mitochondrial changes characteristic of mitochondrial permeability transition. Mice lacking Ppif or Mlkl or given an inhibitor of mitochondrial permeability transition displayed attenuated oxalate-induced AKI. Dual genetic deletion of Ppif and Mlkl or pharmaceutical inhibition of necroptosis was partially redundant, implying interlinked roles of these two pathways of regulated necrosis in acute oxalosis. Similarly, inhibition of mitochondrial permeability transition suppressed crystal-induced cell death in primary human tubular epithelial cells. PPIF and phosphorylated MLKL localized to injured tubules in diagnostic human kidney biopsies of oxalosis-related AKI. CONCLUSIONS: Mitochondrial permeability transition-related regulated necrosis and necroptosis both contribute to oxalate-induced AKI, identifying PPIF as a potential molecular target for renoprotective intervention.Peer reviewe

NAD+ metabolism and therapeutic strategies in cardiovascular diseases

Nicotinamide adenine dinucleotide (NAD+) is a central and pleiotropic metabolite involved in cellular energy metabolism, cell signaling, DNA repair, and protein modifications. Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Metabolic stress and aging directly affect the cardiovascular system. Compelling data suggest that NAD + levels decrease with age, obesity, and hypertension, which are all notable risk factors for CVD. In addition, the therapeutic elevation of NAD + levels reduces chronic low-grade inflammation, reactivates autophagy and mitochondrial biogenesis, and enhances oxidative metabolism in vascular cells of humans and rodents with vascular disorders. In preclinical models, NAD + boosting can also expand the health span, prevent metabolic syndrome, and decrease blood pressure. Moreover, NAD + storage by genetic, pharmacological, or natural dietary NAD + -increasing strategies has recently been shown to be effective in improving the pathophysiology of cardiac and vascular health in different animal models, and human health. Here, we review and discuss NAD + -related mechanisms pivotal for vascular health and summarize recent experimental evidence in NAD + research directly related to vascular disease, including atherosclerosis, and coronary artery disease. Finally, we comparatively assess distinct NAD + precursors for their clinical efficacy and the efficiency of NAD + elevation in the treatment of major CVD. These findings may provide ideas for new therapeutic strategies to prevent and treat CVD in the clinic

Pathophysiology and targeted treatment of cholesterol crystal embolism and the related thrombotic angiopathy

Cholesterol crystal (CC) embolism is a complication of advanced atherosclerotic plaques located in the major arteries. This pathological condition is primarily induced by interventional and surgical procedures or occurs spontaneously. CC can induce a wide range of tissue injuries including CC embolism syndrome, a spontaneous or intervention-induced complication of advanced atherosclerosis, while treatment of CC embolism has remained empiric. Vascular occlusions caused by CC embolism may exceed the ischemia tolerance of many tissues, particularly when small arteries are affected. The main approach to CC embolism is primary prophylaxis in patients at risk by stabilizing atherosclerotic plaques and avoiding unnecessary catheter interventions. During CC embolism, the use of platelet inhibitors to avoid abnormal activation and aggregation and anticoagulants may reduce the risk of vascular occlusions and tissue ischemia. This probably explains the relatively low prevalence of clinical manifestations of CC embolism, which are frequently found in autopsy studies. In this review, we summarized the current knowledge on the pathophysiology of CC embolism syndrome deriving from clinical observations and experimental mouse models. Furthermore, we described the risk factors of CC embolism in humans as well as the experimental studies based on empiric treatments. We also discuss potential therapeutic interventions based on recent experimental data and emerging drug options evolving from other research domains. Given the substantial unmet medical need to improve the outcomes of CC embolism, the identification of effective treatment strategies is urgently needed