Targeting cholesterol crystals in atherosclerosis with cholesterol solubilizing 2-hydroxypropyl-β-cyclodextrin

Atherosclerosis is the underlying pathology of cardiovascular diseases (CVDs), the leading cause of deaths worldwide. Elevated blood cholesterol levels have been linked to this slowly progressing inflammatory disease and lowering the amount of circulating low-density lipoprotein (LDL) cholesterol is one of the most successful treatment approaches. Crystalline cholesterol deposits in the vessel wall are a hallmark of advanced atherosclerotic plaques, but they actually already occur in early stages of atherosclerosis development. Cholesterol crystals (CCs) can activate the NLRP3 inflammasome, a cytosolic multimolecular signaling complex present in innate immune cells, such as macrophages, which are the most prominent cell type in the developing atherosclerotic plaque. NLRP3 inflammasome activation results in the release of the pro-inflammatory cytokines IL-1β and IL-18, which are key contributors to the vascular inflammation driving atherosclerosis progression. Therefore, presumably a reduction in the amount of CCs in atherosclerotic plaques could also decrease vascular inflammation and consequently atherosclerosis progression. In this study, whether 2-hydroxypropyl-β-cyclodextrin (CD), a compound that solubilizes cholesterol and removes cholesterol from cells, is effective in reducing vascular CC deposition or removing CCs from atherosclerotic plaques and thereby preventing atherosclerosis development was investigated in mice. CD treatment indeed impaired murine atherosclerosis development, which was indicated by a reduction of atherosclerotic plaque size and CC load. Moreover, CD treatment even mediated the regression of already established atherosclerotic plaques. The molecular mechanisms of CD-mediated atheroprotection were further examined in vitro in macrophages. CD treatment of CC-loaded macrophages mediated the dissolution of intracellular CCs and subsequently promoted crystal-derived cholesterol esterification, efflux and metabolism to oxysterols such as 27-hydroxycholesterol. Some oxysterols function as endogenous agonists for liver X receptor (LXR) transcription factors, which in turn activate genes regulating cholesterol efflux and antiinflammatory processes. Indeed, CD treatment induced LXR-mediated transcriptional reprogramming of macrophages towards increased cholesterol efflux. Thereby, CD promoted the reverse cholesterol transport and excretion of crystal-derived cholesterol from CC-loaded macrophages in an LXR-dependent manner. Moreover, LXR transcription factor activation was required for the atheroprotective and anti-inflammatory effects of CD in vivo in murine atherosclerosis. This study shows that CD is atheroprotective in mice by dissolving and removing CCs from atherosclerotic plaques and by promoting the production of cholesterol-derived endogenous LXR agonists that activate anti-atherogenic transcriptional processes. Since CD treatment is already approved for clinical use by the U.S. food and drug administration (FDA), it could be directly tested in clinical trials for the prevention or treatment of human atherosclerosis.Cholesterinkristall-Targeting in Atherosklerose mit Cholesterin lösendem 2-Hydroxypropyl-β-cyclodextrin Kardiovaskuläre Erkrankungen sind die häufigste Todesursache weltweit. In den meisten Fällen ist die Atherosklerose, eine progressive, chronische Entzündungskrankheit der Blutgefäße, die pathologische Ursache dieser kardiovaskulären Erkrankungen. Ein erhöhter Cholesterinspiegel im Blut gilt als wichtiger Risikofaktor für die Entstehung der Atherosklerose, und die medikamentöse Senkung von zirkulierendem LDL-Cholesterin ist derzeit eine der erfolgreichsten Behandlungsmethoden. Ablagerungen von kristallinem Cholesterin in den Gefäßwänden gelten als Merkmal fortgeschrittener atherosklerotischer Plaques. Darüberhinaus sind Cholesterinkristalle aber bereits in den frühen Entwicklungsstadien der Atherosklerose vorhanden. Makrophagen, die häufigsten und bedeutendsten Zellen in atherosklerotischen Plaques, erkennen Cholesterinkristalle mithilfe des NLRP3 Inflammasoms. Dieser Multiproteinkomplex des angeborenen Immunsystems verursacht die Ausschüttung der pro-inflammatorischen Zytokine Interleukin (IL)-1β und IL- 18, die entscheidend zur vaskulären Inflammation beitragen und damit für das Voranschreiten der Atherosklerose verantwortlich sind. Eine Reduktion der Cholesterinkristallmenge in atherosklerotischen Plaques könnte voraussichtlich die vaskuläre Inflammation und folglich das Voranschreiten der Atherosklerose verringern. In dieser Studie wurde im Mausmodell untersucht, ob 2-Hydroxypropyl-β-cyclodextrin (CD), welches Cholesterin in Lösung bringt und aus Zellen entfernen kann, auch die vaskuläre Ablagerung von Cholesterinkristallen reduziert oder diese aus den atherosklerotischen Plaques entfernen kann, und ob dadurch die Entwicklung und das Voranschreiten der murinen Atherosklerose verhindert wird. Tatsächlich beeinträchtigte die Behandlung mit CD die Entwicklung der Atherosklerose in Mäusen, was an einer Reduktion der Plaquegröße und der Cholesterinkristallmenge im Plaque erkennbar war. Zudem bewirkte die Behandlung mit CD sogar die Regression von bereits etablierten atherosklerotischen Plaques. Daraufhin wurden die molekularen Mechanismen des atheroprotektiven Effekts von CD in vitro in Makrophagen untersucht. Die Behandlung von Cholesterinkristall-beladenen Makrophagen mit CD führte zur Auflösung der intrazellulären Cholesterinkristalle und förderte die Esterifizierung und den Efflux des aus den Kristallen herausgelösten Cholesterins. Zudem wurde dieses Cholesterin vermehrt zu Oxysterinen, wie zum Beispiel 27-Hydroxycholesterin, metabolisiert. Einige Oxysterine fungieren als endogene Agonisten für LXR (liver X receptor ) Transkriptionsfaktoren, die wiederum Gene aktivieren, welche den Cholesterinefflux und antiinflammatorische Prozesse regulieren. Tatsächlich induzierte die Behandlung mit CD eine LXR-basierte transkriptionelle Umprogrammierung der Makrophagen auf erhöhten Cholesterinefflux. Damit einhergehend förderte CD in Abhängigkeit von LXR den reversen Cholesterintransport und die Ausscheidung von ursprünglich kristallinem Cholesterin aus Cholesterinkristall-beladenen Makrophagen. Darüber hinaus war die Aktivierung von LXR Transkriptionsfaktoren auch für die atheroprotektiven und anti-inflammatorischen Effekte von CD in vivo in muriner Atherosklerose erforderlich. Die vorliegende Studie zeigt eine atheroprotektive Wirkung von CD im Mausmodell, die mit der Auflösung und Beseitigung von Cholesterinkristallen aus atherosklerotischen Plaques einhergeht. Dabei spielt die erhöhte Produktion von Cholesterin-basierten endogenen LXR Agonisten, welche zur Aktivierung anti-atherogener Transkriptionsprozesse führen, eine entscheidende Rolle. Da eine Behandlung mit CD bereits von der FDA (U.S. food and drug administration) für die klinische Nutzung zugelassen ist, könnte CD unmittelbar in klinischen Studien zur Prävention und Behandlung von humaner Atherosklerose getestet werden

CD36 coordinates NLRP3 inflammasome activation by facilitating the intracellular nucleation from soluble to particulate ligands in sterile inflammation

Particulate ligands including cholesterol crystals and amyloid fibrils induce NLRP3-dependent production of interleukin-1β (IL-1β) in atherosclerosis, Alzheimer's disease and diabetes. Soluble endogenous ligands including oxidized-LDL, amyloid-β and amylin peptides accumulate in these diseases. Here we identify a CD36-mediated endocytic pathway that coordinates the intracellular conversion of these soluble ligands to crystals or fibrils, resulting in lysosomal disruption and NLRP3-inflammasome activation. Consequently, macrophages lacking CD36 failed to elicit IL-1β production in response to these ligands and targeting CD36 in atherosclerotic mice reduced serum IL-1β and plaque cholesterol crystal accumulation. Collectively, these findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation

High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3

High Density Lipoprotein (HDL) mediates reverse cholesterol transport and it is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional repressor ATF3, as an HDL-inducible target gene in macrophages that down-regulates the expression of Toll-like receptor (TLR)-induced pro-inflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of novel HDL-based therapies

Cholesterol Crystals and Inflammation

Chronic vascular inflammation is regarded to have a key role in cardiovascular disease. However, the elicitors of this inflammatory response in the vessel wall are currently not well understood. Excessive amounts of cholesterol, an abundant and fundamental lipid molecule in mammalian cells, can trigger the development and progression of atherosclerosis. Accumulation of cholesterol in early atherosclerotic lesions results in the formation of macrophage foam cells, and crystalline cholesterol is found as a hallmark of advanced atherosclerotic plaques. Cholesterol crystals can activate a multimolecular signaling complex of the innate immune system, the NLRP3 inflammasome, resulting in a caspase-1 mediated activation and secretion of proinflammatory interleukin-1 family cytokines. Furthermore, crystalline cholesterol is thought to induce plaque rupture by physical disruption of the fibrous cap covering atherosclerotic lesions. Here we review the role of cholesterol deposition and crystallization for inflammatory responses in cardiovascular diseases

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation

Weekly Treatment of 2-Hydroxypropyl-beta-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice

Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Two-hydroxypropyl-beta-cyclodextrin (HP-B-CD) is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs). Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr(-/-)) mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE) storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7alpha-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.</jats:p