Neutrophil Extracellular Traps Initiate Gallstone Formation.

The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.

Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM.

We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo-scanning electron microscopy (SEM), and cryo-focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media-lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media-lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.

Miliarial-type gout in association with chronic use of glucocorticoids.

A 59-year-old male patient with long-standing tophaceous gout (more than 30 years) characterized by polyarticular involvement and recurrent disseminated tophi formation; his past medical history is relevant for poor adherence to urate-lowering medications, as well as persistent use of self-prescribed systemic glucocorticoids. Despite achieving therapeutic goals for serum uric acid levels, new tophi formation with an intradermal location in the form of "miliarial-type gout" was documented. Due to functional limitations, the patient underwent surgical resection of the olecranon bursa. This case illustrates a widespread and recurrent tophi formation associated with long-standing gout and regular and sustained glucocorticoid use, despite an adequate disease control based on serum urate levels and involving an intradermal location of tophi presenting as "miliarial-type" lesions. In addition, the coexistence of urate and cholesterol crystal deposition disease in olecranon gouty bursitis is presented. Finally, a sonographic extended field of view of lesions distributed along the patient's extremities is presented as a novel characterization of this condition.

Cholesterol crystal formation is a unifying pathogenic mechanism in the development of diabetic retinopathy.

AIMS/HYPOTHESIS: Hyper-reflective crystalline deposits found in retinal lesions have been suggested to predict the progression of diabetic retinopathy, but the nature of these structures remains unknown. METHODS: Scanning electron microscopy and immunohistochemistry were used to identify cholesterol crystals (CCs) in human donor, pig and mouse tissue. The effects of CCs were analysed in bovine retinal endothelial cells in vitro and in db/db mice in vivo using quantitative RT-PCR, bulk RNA sequencing, and cell death and permeability assays. Cholesterol homeostasis was determined using 2H2O and 2H7-cholesterol. RESULTS: We identified hyper-reflective crystalline deposits in human diabetic retina as CCs. Similarly, CCs were found in the retina of a diabetic mouse model and a high-cholesterol diet-fed pig model. Cell culture studies demonstrated that treatment of retinal cells with CCs can recapitulate all major pathogenic mechanisms leading to diabetic retinopathy, including inflammation, cell death and breakdown of the blood-retinal barrier. Fibrates, statins and α-cyclodextrin effectively dissolved CCs present in in vitro models of diabetic retinopathy, and prevented CC-induced endothelial pathology. Treatment of a diabetic mouse model with α-cyclodextrin reduced cholesterol levels and CC formation in the retina, and prevented diabetic retinopathy. CONCLUSIONS/INTERPRETATION: We established that cholesterol accumulation and CC formation are a unifying pathogenic mechanism in the development of diabetic retinopathy.

Evaluation of cholesterol crystals in carotid plaque by dual energy computed tomography.

Cholesterol crystals (CCs) in carotid plaques might be an indicator of vulnerability, although they have not been fully investigated and non-invasive methods of assessment have not been established. This study examines the validity of assessing CCs using dual-energy computed tomography (DECT) that uses X-rays with different tube voltages for imaging, allowing material discrimination. We retrospectively evaluated patients who had undergone preoperative cervical computed tomography angiography and carotid endarterectomy between December 2019 and July 2020. We developed CC-based material decomposition images (MDIs) by scanning CCs crystallized in the laboratory using DECT. We compared the percentage of CCs in stained slides defined by cholesterol clefts with the percentage of CCs displayed by CC-based MDIs. Thirty-seven pathological sections were obtained from 12 patients. Thirty-two sections had CCs; of these, 30 had CCs on CC-based MDIs. CC-based MDIs and pathological specimens showed a strong correlation. Thus, DECT allows the evaluation of CCs in carotid artery plaques.

Lipids, hyperreflective crystalline deposits and diabetic retinopathy: potential systemic and retinal-specific effect of lipid-lowering therapies.

The metabolically active retina obtains essential lipids by endogenous biosynthesis and from the systemic circulation. Clinical studies provide limited and sometimes conflicting evidence as to the relationships between circulating lipid levels and the development and progression of diabetic retinopathy in people with diabetes. Cardiovascular-system-focused clinical trials that also evaluated some retinal outcomes demonstrate the potential protective power of lipid-lowering therapies in diabetic retinopathy and some trials with ocular primary endpoints are in progress. Although triacylglycerol-lowering therapies with fibrates afforded some protection against diabetic retinopathy, the effect was independent of changes in traditional blood lipid classes. While systemic LDL-cholesterol lowering with statins did not afford protection against diabetic retinopathy in most clinical trials, and none of the trials focused on retinopathy as the main outcome, data from very large database studies suggest the possible effectiveness of statins. Potential challenges in these studies are discussed, including lipid-independent effects of fibrates and statins, modified lipoproteins and retinal-specific effects of lipid-lowering drugs. Dysregulation of retinal-specific cholesterol metabolism leading to retinal cholesterol accumulation and potential formation of cholesterol crystals are also addressed.

Protective effect of SIRT6 on cholesterol crystal-induced endothelial dysfunction via regulating ACE2 expression.

Sirtuins are a family of highly conserved nicotinamide adenine dinucleotide (NAD+)-dependent enzymes. Among the sirtuins, SIRT1 and SIRT6 participate in the regulation of endothelial functions and play significant roles in the physiological and pathological processes of cardiovascular diseases (CVD). Recently, our study found that minute cholesterol crystals (CC) can be endocytosed by endothelial cells and further impair endothelial functions. Since previous studies have reported that angiotensin-converting enzyme (ACE2) involves Angiotensin (Ang) II-induced inflammation in endothelial cells, this study was designed to investigate the role of SIRT1 and SIRT6 in CC-induced variation of ACE2 expression and the related mechanism between SIRT6 and ACE2. We found that ACE2 is involved in CC-induced endothelial dysfunction, which inhibits decreases in nitric oxide (NO) level and endothelial nitric oxide synthase (eNOS) activity and increases in inflammatory factors and adhesion molecules. Besides, SIRT1 and SIRT6 regulated the protein expression of ACE2 in CC-stimulated human umbilical vein endothelial cells (HUVECs). Moreover, bioinformatics analysis from the Enrichr database indicated that activating transcription factor 2 (ATF2), is highly correlated with genes that significantly upregulated after infection with the SIRT6 adenovirus vector. In CC-induced HUVECs, ACE2 expression was up-regulated in cells transfected with ATF2 siRNA. However, further mechanism studies revealed that overexpression of SIRT6 decreases the accumulation of p-ATF2 in the nucleus, but did not affect p-ATF2 expression in the cytoplasm. Taken together, these data indicated that SIRT6 regulates ACE2 might via inhibiting the accumulation of nucleus p-ATF2 in CC-induced endothelial dysfunction.

Poly-ß-cyclodextrin Supramolecular Nanoassembly with a pH-Sensitive Switch Removing Lysosomal Cholesterol Crystals for Antiatherosclerosis.

Cholesterol crystals (CCs), originally accumulating in the lysosome of cholesterol-laden cells, can aggravate the progression of atherosclerosis. ß-cyclodextrin (CD) is a potent cholesterol acceptor or CC solubilizer. However, the random extraction of cholesterol impedes the in vivo application of CD for removing lysosomal CCs. Here, we exploit poly-ß-cyclodextrin (pCD) as a lysosomal CC solubilizer and dextran sulfate grafted with benzimidazole (BM) as a pH-sensitive switch (pBM) to self-assemble into a supramolecular nanoassembly (pCD/pBM-SNA). The CD cavity in pCD/pBM-SNA can be efficiently sealed by hydrophobic BM at pH 7.4 (OFF). After it enters the lysosome, pCD/pBM-SNA disassembles, recovers the CD cavity to dissolve CCs into free cholesterol due to the protonation of BM (ON), and reduces CCs, finally enhancing the cholesterol efflux and promoting atherosclerosis regression. Our findings provide an "OFF-ON" tactic to remove lysosomal CCs for antiatherosclerosis as well as other diseases such as Niemann-Pick type C diseases with excessive cholesterol accumulation in the lysosome.

Pyroptosis is a critical immune-inflammatory response involved in atherosclerosis.

Pyroptosis is a form of programmed cell death activated by various stimuli and is characterized by inflammasome assembly, membrane pore formation, and the secretion of inflammatory cytokines (IL-1ß and IL-18). Atherosclerosis-related risk factors, including oxidized low-density lipoprotein (ox-LDL) and cholesterol crystals, have been shown to promote pyroptosis through several mechanisms that involve ion flux, ROS, endoplasmic reticulum stress, mitochondrial dysfunction, lysosomal rupture, Golgi function, autophagy, noncoding RNAs, post-translational modifications, and the expression of related molecules. Pyroptosis of endothelial cells, macrophages, and smooth muscle cells in the vascular wall can induce plaque instability and accelerate atherosclerosis progression. In this review, we focus on the pathogenesis, influence, and therapy of pyroptosis in atherosclerosis and provide novel ideas for suppressing pyroptosis and the progression of atherosclerosis.

SIRT6 inhibits cholesterol crystal-induced vascular endothelial dysfunction via Nrf2 activation.

Sirtuin 6 (SIRT6), a nicotinamide adenine dinucleotide-dependent deacetylase, participates in various age-related disorders, such as dyslipidemia and cardiovascular diseases. Recent studies have revealed that minute cholesterol crystals (CCs), which are generated after excess free cholesterol accumulation, form not only in mature atherosclerotic plaques but also extremely early in atherosclerosis. Since endothelial dysfunction is an early feature of atherogenesis, this study was designed to investigate the role of SIRT6 in minute CC-induced endothelial dysfunction and the related mechanism. We found that minute CCs could be endocytosed by endothelial cells (ECs), which then decreased nitric oxide (NO) levels and endothelial nitric oxide synthase (eNOS) activity and expression, upregulated the expression of adhesion molecules and enhanced monocyte adhesion to ECs. In addition, minute CCs significantly suppressed SIRT6 expression in ECs. Moreover, the overexpression of SIRT6 could mitigate minute CC-induced endothelial dysfunction. In addition, the expression of Nuclear factor erythroid2-related factor2 (Nrf2) was suppressed after minute CC treatment, whereas SIRT6 overexpression reversed this decrease in Nrf2 expression. More importantly, Nrf2 activation also notably attenuated minute CC-induced endothelial dysfunction. In vivo experiments further indicated that endothelium-specific SIRT6 depletion impaired vascular endothelial function and suppressed Nrf2 expression in hyperlipidemic mice. Taken together, these results indicate that SIRT6 rescues minute CC-induced endothelial dysfunction partly via Nrf2 activation.