Cholesterol Crystal Formation Research Articles

Cholelithiasis, Gut Microbiota and Bile Acids after Bariatric Surgery—Can Cholelithiasis Be Prevented by Modulating the Microbiota? A Literature Review

Background: Cholelithiasis is one of the more common complications following bariatric surgery. This may be related to the rapid weight loss during this period, although the exact mechanism of gallstone formation after bariatric surgery has not been fully elucidated. Methods: The present literature review focuses on risk factors, prevention options and the impact of the gut microbiota on the development of gallbladder stones after bariatric surgery. Results: A potential risk factor for the development of cholelithiasis after bariatric surgery may be changes in the composition of the intestinal microbiota and bile acids. One of the bile acids—ursodeoxycholic acid—is considered to reduce the concentration of mucin proteins and thus contribute to reducing the formation of cholesterol crystals in patients with cholelithiasis. Additionally, it reduces the risk of both asymptomatic and symptomatic gallstones after bariatric surgery. Patients who developed gallstones after bariatric surgery had a higher abundance of Ruminococcus gnavus and those who did not develop cholelithiasis had a higher abundance of Lactobacillaceae and Enterobacteriaceae. Conclusion: The exact mechanism of gallstone formation after bariatric surgery has not yet been clarified. Research suggests that the intestinal microbiota and bile acids may have an important role in this.

Sterol O-acyltransferase (SOAT/ACAT) activity is required to form cholesterol crystals in hepatocyte lipid droplets

ObjectiveExcess cholesterol storage can induce the formation of cholesterol crystals in hepatocyte lipid droplets. Such crystals distinguish metabolic dysfunction associated steatohepatitis (MASH) from simple steatosis and may underlie its pathogenesis by causing cell damage that triggers liver inflammation. The mechanism linking cholesterol excess to its crystallization in lipid droplets is unclear. As cholesteryl esters localize to and accumulate in lipid droplets more readily than unesterified free cholesterol, we investigated whether cholesterol esterification by sterol O-acyltransferase (SOAT), also known as acyl co-A cholesterol acyltransferase (ACAT), is required for hepatocyte lipid droplet crystal formation. MethodCholesterol crystals were measured in cholesterol loaded Hep3B hepatocytes, RAW264.7 macrophages, and mouse liver using polarizing light microscopy. We examined the effect of blocking SOAT activity on crystal formation and compared these results to features of cholesterol metabolism and the progression to intracellular crystal deposits. ResultsCholesterol loading of Hep3B cells caused robust levels of lipid droplet localized crystal formation in a dose- and time-dependent manner. Co-treatment with SOAT inhibitors and genetic ablation of SOAT1 blocked crystal formation. SOAT inhibitor also blocked crystal formation in low density lipoprotein (LDL) treated Hep3B cells, acetylated LDL treated RAW 264.7 macrophages, and in the liver of mice genetically predisposed to hepatic cholesterol overload and in mice with cholesterol enriched diet-induced MASH. ConclusionSOAT1-mediated esterification may underlie cholesterol crystals associated with MASH by concentrating it in lipid droplets. These findings imply that inhibiting hepatocyte SOAT1 may be able to alleviate cholesterol associated MASH. Moreover, that either a lipid droplet localized cholesteryl ester hydrolase is required for cholesterol crystal formation, or the crystals are composed of cholesteryl ester.

Transcriptomic analysis reveals the critical role of chemokine signaling in the anti-atherosclerosis effect of Xuefu Zhuyu decoction

Ethnopharmacological relevanceThe process of atherosclerosis (AS) is complicated. Transcriptomics technology can assist in discovering the underlying mechanisms and exploring the key targets of Traditional Chinese Medicine (TCM) against atherosclerosis. AimThis study aimed to investigate targets and signaling pathways significantly related to AS and the potential intervention targets of Xuefu Zhuyu decoction by transcriptomics. Materials and methodsAS models were established by subjecting ApoE-/-mice to an 8-week high-fat diet. Structural changes and plaque formation in the aortic root were observed using hematoxylin-eosin staining (HE staining), while Oil Red O staining was employed to visualize lipid deposition within the aortic root plaque. Movat staining and immunohistochemical staining were conducted to examine the components present in the aortic root plaque. Macrophage content within the plaque was observed through immunofluorescence. Additionally, mRNA sequencing was performed on aortic tissues to identify differentially expressed genes. Enrichment analysis was performed using GO and KEGG analysis. Visualization of the protein-protein interaction (PPI) network was achieved using Cytoscape 3.7.1 and STRING. Western blotting (WB) was employed to assess the protein expression of major differentially expressed genes in the aortic tissue. The drug freeze-dried powder of Xuefu Zhuyu decoction was prepared and the RAW264.7 cells were induced by lipopolysaccharide (LPS) to build an in vitro model. Real-time quantitative PCR was employed to measure the mRNA expression of major differential genes. ResultsAfter ApoE-/- mice were fed with an 8-week high-fat diet, observable changes included the thinning of the aortic root wall, the accumulation of foam cells within the plaque, and the formation of cholesterol crystals in the model group. Treatment with Xuefu Zhuyu (XFZY) decoction for 12 weeks significantly reduced the lipid deposition and the number of macrophages (P < 0.05) and significantly increased the collagen content within the plaque (P < 0.01). Enrichment analysis revealed a high enrichment of the Cytokine-cytokine receptor interaction pathway and Chemokine signaling pathway. Noteworthy genes involved in this response included Ccl12, Ccl22, Cx3cr1, Ccr7, Ccr2, Tnfrsf25, and Gdf5. Xuefu Zhuyu decoction significantly downregulated the expression of CX3CL1 and CX3CR1 (P < 0.05) and upregulated the expression of GDF5 (P < 0.01). Compared with control group, in cell models, the mRNA expressions of Ccl12, Ccl22, and Ccr2 were significantly upregulated (P < 0.05 or P < 0.01). Xuefu Zhuyu decoction significantly downregulated the expression of Ccl12, Ccl22, Cx3cr1, Ccr7 and Ccr2 (P < 0.05 or P < 0.01). ConclusionXuefu Zhuyu decoction demonstrates effective regulation of plaque components, retarding plaque progression and preserving plaque stability by modulating lipid metabolism and inflammatory responses. Subsequent transcriptome analysis identified the Cytokine-cytokine receptor interaction and Chemokine signaling pathway as potential key pathways for the therapeutic effects of Xuefu Zhuyu decoction. This insight not only provides crucial avenues for further exploration into the mechanisms underlying Xuefu Zhuyu decoction but also offers valuable perspectives and hypotheses for enhancing disease prevention and treatment strategies.

Vascular smooth muscle cells in response to cholesterol crystals modulates inflammatory cytokines release and promotes neutrophil extracellular trap formation

BackgroundThe formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response.MethodsHuman aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy.ResultsVSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K–AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation.ConclusionWe demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

The effect of colchicine on cholesterol crystal formation, expansion and morphology: a potential mechanism in atherosclerosis.

Inflammation is pivotal to the progression of atherosclerosis. Cholesterol crystals (CCs) that grow and enlarge within the plaque core can cause plaque rupture and trigger inflammation as they deposit into the atherosclerotic bed. Thus, agents that affect CC formation, expansion, and morphology may reduce cardiovascular (CV) risk independent of lipid-lowering and anti-inflammatory therapy. Because colchicine is highly concentrated in leukocytes that can enter the atherosclerotic plaque core, we tested its effect on the formation and growth of CCs in bench experiments to determine whether it may have direct effects on CCs, independent of its known anti-inflammatory actions. Different dosages of colchicine mixed with cholesterol (0.05-5 mg/ml/g of cholesterol) were used to influence the formation CCs and volume expansion in vitro. These were compared to control samples with cholesterol in ddH2O without colchicine. In an ex vivo study, fresh atherosclerotic human plaques were incubated with and without colchicine in a water bath at 37°C for 48 h to assess the impact of colchicine on CC morphology. Scanning electron microscopy (SEM) was utilized to analyze CC morphology in samples from the various treatment groups. The addition of colchicine to cholesterol caused a substantial dose-dependent reduction in volume (p < 0.05). Pairwise comparisons of volume reduction, showed a significant reduction in volume at 5 mg/ml/g when compared to control (p < 0.02) but the calculated Cohen's d effect size was large for five of the six pairwise comparisons. By SEM, CCs from both in vitro and ex vivo samples treated with colchicine had evidence of dissolution and changes in their morphology as evidenced by the loss of their sharp edges. In contrast, CCs in untreated specimens retained their typical geometric structure. Colchicine can reduce CC formation and expansion and alter CC morphology. These previously unappreciated effects of colchicine may contribute to its clinical benefit in patients with CV disease independent of its anti-inflammatory effects.

Cholesterol Crystals in Odontogenic Cysts: An In-Depth Narrative of Pathogenesis

Cholesterol crystals (CCs) are a relatively common finding in infected odontogenic cysts occurring in about 60% of the cases. Histopathologically, these appear as longitudinal cleft-like spaces or may appear as a collection in cholesterol granulomata. The occurrence of CCs in odontogenic cysts is closely associated with the pathophysiology of the lesions. Despite being fairly common, not much is known about the sources of cholesterol, the mechanism of formation of these clefts, their pathogenesis, and their clinical implications. The present article focuses on describing CCs with respect to their contents, pathogenesis, and clinical implications. Our review describes the mechanism of the formation of CCs in a detailed manner along with a pictorial depiction of the cascade of events involved in the process. The formation of cholesterol granulomata is a cumulative effect of the breakdown of erythrocytes and leukocytes, intramural hemorrhage, decreased blood supply and lymphatic drainage, hypoxic conditions, increased vascular permeability, accumulation and rupture of lipid-laden macrophages, and foreign body reaction. It would be of great interest to pathologists to have an in-depth understanding of the pathogenesis, nature, and implications of CCs, which are very common yet enigmatic entities.

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

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. 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. 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. We established that cholesterol accumulation and CC formation are a unifying pathogenic mechanism in the development of diabetic retinopathy.

Atorvastatin Attenuates Diet-Induced Non-Alcoholic Steatohepatitis in APOE*3-Leiden Mice by Reducing Hepatic Inflammation.

Patients with metabolic syndrome are often prescribed statins to prevent the development of cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma cholesterol lowering (-30%) and anti-atherogenic effects (severe lesion size -48%), atorvastatin significantly reduced hepatic steatosis (-22%), the number of aggregated inflammatory cells in the liver (-80%) and hepatic fibrosis (-92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated mice showed less immunohistochemically stained areas of inflammation markers. Atorvastatin prevented accumulation of free cholesterol in the form of cholesterol crystals (-78%). Cholesterol crystals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its activation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1β (-61%) and IL-18 (-26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crystal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in dyslipidemic patients.

Clinical Images: Cholesterol crystals bursitis.

The patient, a 45-year-old woman, was referred to the outpatient clinic with an effusion of her right olecranon bursa. She had a history of systemic sclerosis, liver cirrhosis, and autoimmune hepatitis that was treated with mycophenolate mofetil and prednisone. About 3 years ago, she started to develop an effusion of the right olecranon bursa. More recently she noticed white skin nodules, which turned out to be calcinosis cutis. There were no signs of arthritis on either clinical examination or imaging. An ultrasound of the right olecranon bursa was performed and revealed a tissue mass with hyperechogenic lesions within, which is compatible with crystal depositions (A). Analysis of the aspirate with the polarization microscope indicated the presence of cholesterol crystals (B; left: original magnification × 40; right: original magnification × 10). These crystals usually look like plates of glass, sometimes with a notch out of a corner. They are variably and mildly birefringent. Under polarized light, they may exhibit a stained-glass effect. Most cases of cholesterol crystals are associated with hyperlipidemia, yet our patient had normal lipoprotein levels and did not use cholesterol-lowering medication. Instead, increased local cell degeneration and/or increased lipid permeability in response to chronic inflammation might have contributed to the formation of cholesterol crystals in the olecranon bursa of our patient. Cholesterol crystals have been described previously just once in a patient with scleroderma, in the subcutis, mimicking calcinosis cutis (1). After failing local corticosteroid injections, the patient was finally referred to the orthopedic surgeon for surgical synovectomy. Cholesterol crystals in bursal fluid of a patient with scleroderma has not been previously reported, as most of the cases reported so far in patients with rheumatic diseases are concerning patients with rheumatoid arthritis (2). Disclosure Form Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Impact of small dense low-density lipoprotein cholesterol on cholesterol crystals in patients with acute coronary syndrome: An optical coherence tomography study

The presence of cholesterol crystals (CCs) is recognized as a component of vulnerable atherosclerotic plaques at risk of rupture. The phagocytosis of atherogenic lipid factors by macrophages precedes and promotes the formation of vulnerable plaques, but it is not clear how these factors affect the formation of CC. This study aimed to evaluate the relationship between lipid biomarkers such as small dense low-density lipoprotein cholesterol (sd-LDL-c) and CC detected by optical coherence tomography (OCT) in patients with acute coronary syndrome (ACS). Serum samples were collected immediately before coronary angiography in consecutive 174 patients with ACS who did not take statins and underwent OCT imaging of the culprit lesion. The sd-LDL-c levels were measured using a direct homogenous assay. CC was defined as a thin linear structure with high reflectivity and low signal attenuation on the OCT images. CC was identified in 85 patients (48.9%). The prevalence of CC was significantly higher in lesions with ruptured plaques and greater macrophage grade. The sd-LDL-c levels were significantly higher in the patients with CC (41.6 vs. 31.2 mg/dL, p=0.01) although there were no significant differences in the levels of LDL-c and apolipoprotein B. The CC group also had higher levels of apolipoprotein C3 and HbA1c levels. In multiple logistic regression analysis, sd-LDL-c was an independent risk factor of CC (odds ratio, 1.19 per 10 mg/dL; p=0.03). sd-LDL may play an important role in the presence of CC in patients with ACS.

Discovery of an insulin-induced gene binding compound that ameliorates nonalcoholic steatohepatitis by inhibiting sterol regulatory element-binding protein-mediated lipogenesis.

NASH is associated with high levels of cholesterol and triglyceride (TG) in the liver; however, there is still no approved pharmacological therapy. Synthesis of cholesterol and TG is controlled by sterol regulatory element-binding protein (SREBP), which is found to be abnormally activated in NASH patients. We aim to discover small molecules for treating NASH by inhibiting the SREBP pathway. Here, we identify a potent SREBP inhibitor, 25-hydroxylanosterol (25-HL). 25-HL binds to insulin-induced gene (INSIG) proteins, stimulates the interaction between INSIG and SCAP, and retains them in the endoplasmic reticulum, thereby suppressing SREBP activation and inhibiting lipogenesis. In NASH mouse models, 25-HL lowers levels of cholesterol and TG in serum and the liver, enhances energy expenditure to prevent obesity, and improves insulin sensitivity. 25-HL dramatically ameliorates hepatic steatosis, inflammation, ballooning, and fibrosis through down-regulating the expression of lipogenic genes. Furthermore, 25-HL exhibits both prophylactic and therapeutic efficacies of alleviating NASH and atherosclerosis in amylin liver NASH model diet-treated Ldlr-/- mice, and reduces the formation of cholesterol crystals and associated crown-like structures of Kupffer cells. Notably, 25-HL lowers lipid contents in serum and the liver to a greater extent than lovastatin or obeticholic acid. 25-HL shows a good safety and pharmacokinetics profile. This study provides the proof of concept that inhibiting SREBP activation by targeting INSIG to lower lipids could be a promising strategy for treating NASH. It suggests the translational potential of 25-HL in human NASH and demonstrates the critical role of SREBP-controlled lipogenesis in the progression of NASH by pharmacological inhibition.

TO STUDY THE ASSOSIATION BETWEEN SERUM IRON AND FERRITIN LEVEL WITH GALLSTONE DISEASE

INTRODUCTION: Gallstone disease is a common clinical condition affecting the adult population of both sexes. Iron deciency was found to be a new parameter in aetiology of gallstones[5, 6,7]. Iron deciency has been shown to alter the activity of several hepatic enzymes, leading to increased gallbladder cholesterol saturation and promotion of cholesterol crystal formation[8, 9]. Iron acts as a coenzyme for nitric oxide synthetase (NOS), which synthesizes nitric oxide (NO) important for the maintenance of gallbladder tone and normal relaxation [10, 11]. METHOD: Prospective case control study was conducted In Dr.B.R.A.M. Hospital, Raipur On January 2020 to December 2020. sample size was for case is 60 and for control 60 taken On the basis of inclusion and exclusion criteria . patient sonological conrmed and estimate the serum iron and ferritin level . Data are analyzed with descriptive statistical principals RESULT: 31.67% patient of cholelithiasis have serum iron level below normal value .In control group 21.67% participants have normal serum iron level ( P value = 0.21) 33.33% cases have serum ferritin level below normal range and18.33% of control group have serum ferritin level below normal range ( P value = 0.06 ) CONCLUSION: gallstone are more prevalent in female population than males . low serum iron was not signicantly found in both case and control group with relation to cholelithiasis. low serum ferritin level not signicantly found both in case and control group

Niacin promotes the efflux of lysosomal cholesterol from macrophages via the CD38/NAADP signaling pathway.

The accumulation of free cholesterol in macrophage lysosomes significantly enhances atherogenesis. Our recent study demonstrated that the cluster of differentiation 38 (CD38)/nicotinic acid adenine dinucleotide phosphate (NAADP)/Ca2+ signaling pathway plays a critical role in the efflux of lysosomal free cholesterol from macrophages in atherosclerosis. Niacin, known as nicotinic acid, is one of the oldest lipid-lowering medications showing unique anti-atherosclerotic activity. However, it is unknown whether this anti-atherosclerosis activity is associated with the efflux of lysosomal compartmentalized cholesterol in macrophages. In this study, we investigated the anti-atherosclerotic effects of niacin on the reduction of lysosomal free cholesterol via CD38/NAADP signaling in macrophages derived from low-density lipoprotein receptor (LDLr-/-) mice. Fluorescent filipin and Nile red labeling coupled with confocal microscopy demonstrated that niacin reduced free cholesterol accumulation in lysosomes in a concentration-dependent manner. Transmission electron microscopy also showed that niacin markedly decreased cholesterol crystal formation in lysosomes in oxidized LDL-containing LDLr-/- bone marrow-derived macrophages. Enzyme-linked immunosorbent assays showed that niacin increased NAADP production in a concentration-dependent manner, which was inhibited by small interfering RNA interference of CD38. Therefore, niacin may promote the efflux of lysosomal cholesterol from macrophages via the CD38/NAADP signaling pathway.

Modified Lipoproteins Induce Arterial Wall Inflammation During Atherogenesis.

Circulating apolipoprotein B-containing lipoproteins, notably the low-density lipoproteins, enter the inner layer of the arterial wall, the intima, where a fraction of them is retained and modified by proteases, lipases, and oxidizing agents and enzymes. The modified lipoproteins and various modification products, such as fatty acids, ceramides, lysophospholipids, and oxidized lipids induce inflammatory reactions in the macrophages and the covering endothelial cells, initiating an increased leukocyte diapedesis. Lipolysis of the lipoproteins also induces the formation of cholesterol crystals with strong proinflammatory properties. Modified and aggregated lipoproteins, cholesterol crystals, and lipoproteins isolated from human atherosclerotic lesions, all can activate macrophages and thereby induce the secretion of proinflammatory cytokines, chemokines, and enzymes. The extent of lipoprotein retention, modification, and aggregation have been shown to depend largely on differences in the composition of the circulating lipoprotein particles. These properties can be modified by pharmacological means, and thereby provide opportunities for clinical interventions regarding the prevention and treatment of atherosclerotic vascular diseases.

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.

Abstract 14059: Lycopene Reduces Formation of Cholesterol Crystals and Accelerates Their Dissolution

Introduction: Cholesterol crystals are one of the main culprits behind the rupture of atherosclerotic plaque and resultant thrombosis, the process responsible for the development of heart attack and ischemic stroke, the world’s leading causes of mortality. Today there are no drugs or treatment able to target these crystals. Hypothesis/Objectives and Methods: Effect of lycopene on the growth of cholesterol crystals was investigated. A study was also made on the effect of this carotenoid on the dissolution of already formed cholesterol crystals in vitro and in human atherosclerotic plaques. Results: It was found that lycopene was directly able to disrupt, slow down and facilitate the reversal of the cholesterol crystallisation process. In in vitro experiments, lycopene delayed the time of the beginning of the process by 8 fold and its rate of this process itself by 5 fold. In addition it was able to reduce the cholesterol mass involved into crystallisation by 60%, fig. 1. Even when the cholesterol crystals were formed in the presence of lycopene their size was significantly smaller than when the crystals grew undisrupted, fig. 2. This effect could be observed in a ratio of 1 molecule of lycopene to 1,000-10,000 molecules of cholesterol, which indicates that these two molecules can create a thermodynamically favourable complex, which triggers a long-range ordering transition in their folding. Conclusion: The presented findings provide a sound rationale for future studies of lycopene on its potential prevention of cholesterol crystal formation and facilitation of their disassembly in patients with clinical complications of atherosclerosis.

Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results.

Since its inception more than thirty years ago, electroformation has become the most commonly used method for growing giant unilamellar vesicles (GUVs). Although the method seems quite straightforward at first, researchers must consider the interplay of a large number of parameters, different lipid compositions, and internal solutions in order to avoid artifactual results or reproducibility problems. These issues motivated us to write a short review of the most recent methodological developments and possible pitfalls. Additionally, since traditional manual analysis can lead to biased results, we have included a discussion on methods for automatic analysis of GUVs. Finally, we discuss possible improvements in the preparation of GUVs containing high cholesterol contents in order to avoid the formation of artifactual cholesterol crystals. We intend this review to be a reference for those trying to decide what parameters to use as well as an overview providing insight into problems not yet addressed or solved.

Formation and Cellular Impact of Cholesterol Crystals in Health and Disease.

Cholesterol crystals (CCs) were first discovered in atherosclerotic plaque tissue in the early 1900 and have since been observed and implicated in many diseases and conditions, including myocardial infarction, abdominal aortic aneurism, kidney disease, ocular diseases, and even central nervous system anomalies. Despite the widespread involvement of CCs in many pathologies, the mechanisms involved in their formation and their role in various diseases are still not fully understood. Current knowledge concerning the formation of CCs, as well as the molecular pathways activated upon cellular exposure to CCs, will be explored in this review. As CC formation is tightly associated with lipid metabolism, the role of cellular lipid homeostasis in the formation of CCs is highlighted, including the role of lysosomes. In addition, cellular pathways and processes known to be affected by CCs are described. In particular, CC-induced activation of the inflammasome and production of reactive oxygen species, along with the role of CCs in complement-mediated inflammation is discussed. Moreover, the clinical manifestation of embolized CCs is described with a focus on renal and skin diseases associated with CC embolism. Lastly, potential therapeutic measures that target either the formation of CCs or their impact on different cell types and tissues are highlighted.

Abstract MP49: Macrophage-mediated Extracellular Digestive Exophagy Of Aggregated LDL Is Responsible For The Formation Of Cholesterol Crystals In Atherosclerotic Plaques

Atherosclerosis is a disease that develops overtime through dyslipidemia and chronic inflammation. Formation of cholesterol crystals in the atherosclerotic plaques is a major contributor to the inflammatory response. However, how these crystals form in the atherosclerotic plaques remained poorly understood. We have observed that macrophage-mediated extracellular digestive exophagy of aggregated LDL releases a large amount of free cholesterol in the extracellular space. Therefore, we hypothesized that this release of cholesterol may play a major role in formation of cholesterol crystals in plaques. We have shown previously that TLR4 knockout in macrophages compromises digestive exophagy of aggregated LDL. Thus, to evaluate the role of digestive exophagy in cholesterol crystal formation, we examined cryosections of aortic plaques from Ldlr -/- mice that were gamma-irradiated and reconstituted with WT or Tlr4 -/- bone marrows. Interestingly, TLR4 KO in macrophages reduce both free cholesterol and cholesterol crystal in plaques compared to WT macrophages, suggesting a role of digestive exophagy in cholesterol crystal formation. To investigate whether digestive exophagy of aggregated LDL results in inflammatory response, we cultured bone-marrow derived macrophages expressing the inflammasome marker ASC-citrine with aggregated LDL over time. We observed a gradual increase in number of inflammasomes over a 24 h time period. We also observed extracellular cholesterol crystal formation as early as 2 h after the addition of aggregated LDL. Moreover, treating bone marrow-derived macrophages with acetylated LDL did not result in the formation of extracellular cholesterol crystals, suggesting this process is specific to a subset of modified LDL in the vessel wall. Overall, our data provide evidence that macrophage-mediated digestive exophagy of aggregated LDL is a major contributor to the formation of extracellular cholesterol crystal found in atherosclerotic lesions.

RADIOGRAPHIC FINDINGS OF DENTIGEROUS CYSTS WITH CHOLESTEROL GRANULOMA

<h3>BACKGROUND</h3> Dentigerous cysts are common benign odontogenic cysts.¹ A dentigerous cyst is characterized by an epithelium attachment that is associated with the crown of an unerupted tooth and appears radiographically as a well-defined radiolucency.^2,3 The dentigerous cyst may be a cholesterol granuloma histopathologically when it is associated with numerous cholesterol clefts with foam cells, foreign body giant cells, and macrophages filled with hemosiderin.⁴ Cholesterol granuloma often occurs in the middle ear and may be found in other sites.⁵ There are very few reported cases of cholesterol granuloma in the jaws. <h3>CASE REPORT</h3> We present 4 cases of dentigerous cysts with cholesterol granuloma. All 3 lesions were in the mandible. The first patient was a 34-year-old man who had a lesion in his right mandibular ramus. The second patient was a 45-year-old man with a lesion in the left posterior mandible. The third patient was a 77-year-old man with 2 lesions in the posterior mandible bilaterally. The lesions were mostly well defined. Three were unilocular and one multilocular with sclerotic margins, suggestive of peripheral bone reaction to a possible inflammatory process. The histopathologic diagnosis of these cases was dentigerous cyst with cholesterol granuloma. <h3>DISCUSSION/CONCLUSIONS</h3> The pathogenesis of cholesterol granuloma is still unclear.⁴ Cholesterol granuloma may form because of either possible disruption of ventilation and/or improper drainage and hemorrhage into a closed cavity.⁵ Cholesterol granuloma could lead to the expansion of the lesion and may have a leading role in cyst growth. The formation of large cholesterol crystals causes a foreign body reaction that releases macrophages and multinucleated giant cells. The cholesterol granulomas resist the action of phagocytes and continue to release inflammatory and bone resorptive products that may lead to expansion along with aggressive radiographic features.