動脈硬化壁に蓄積した脂質球の化学的組成について

Increased immunoreactivity of endothelin-1 and endothelin B receptor in human atherosclerotic lesions. A possible role in atherogenesis

In recent years, it has become increasingly clear that atherosclerotic lesions do not occur randomly in the arterial system. Rather, they have been shown to favor certain regions, while sparing others, both in the human (McGill, 1968a; McGill, 1968b; Mitchell and Schwartz, 1965) and in experimental models (Faggiotto and Ross, 1984; Gerrity et al., 1979; Jerome and Lewis, 1985; Joris et al., 1983). Modern computer-assisted image processing techniques have been developed to express the topographic distribution of lesions in statistical terms, thus quantifying precisely previous qualitative studies which have indicated that inflow tracts of ostia and flow dividers are particularly susceptible to early lesion development (Cornhill et al., 1985). Other studies have clarified the cellular composition of atherosclerotic plaques in both man (Geer et al., 1961; Ross et al., 1984) and animal models (Faggiotto and Ross, 1984; Gerrity et al., 1979, Jerome and Lewis, 1985; Joris et al., 1983; Faggiotto et al., 1984; Gerrity, 1981a; Gerrity, 1981b; Jerome and Lewis, 1984; Wissler and Vesselinovitch, 1977), and have provided insight into the relationships between cellular composition and lesion type, location, and progression. The fatty streak lesion (the earliest detectable form of atherosclerosis in man), is characteristically a lipid-rich, flat lesion consisting predominantly of macrophage foam cells with minimal smooth muscle cell involvement (McGill, 1968a). Advanced atherosclerosis in older individuals is more typified by various forms of fibrous plaques dominated by smooth muscle cells and enhanced amounts of connective tissue protein and matrix. The central core of such lesions consists of both intra- and extra-cellular lipid accumulations covered on the luminal aspect by a fibrous cap of smooth muscle and connective tissue. The question of whether the fatty streak is a precursor to the fibrous plaque has long been debated. However, recent data, reviewed by McGill (1968) supports the precursor concept, in that increased surface area coverage by fatty streaking precedes advanced plaques in coronary arteries. Stary (1985) has also shown that lesions in young children consist predominantly of macrophage-derived foam cells, with a few underlying lipid-laden smooth muscle cells. However, he also demonstrated advanced fibrous plaques at the same anatomical sites in older individuals, confirming earlier data (Geer et al., 1961; Robertson et al., 1963) which indicated an age-dependent relationship between fatty streaks and fibrous plaques at the same anatomical site. If, as these data would suggest, the fatty streak is indeed the precursor of the clinically-significant fibrous plaque, then the mechanisms controlling monocyte involvement in fatty streak initiation are of considerable importance, since the monocyte-derived foam cell is the predominant cell of the fatty streak. Furthermore, it has been demonstrated that one of the earliest events to occur in such lesion-susceptible areas is the large-scale recruitment of blood monocytes into the intima (Faggiotto and Ross, 1984; Gerrity et al., 1981a,b; Jerome and Lewis, 1985; Joris et al., 1983; Jerome and Lewis, 1984; Robertson et al., 1963).

Objective: Human atherosclerotic lesions of different stages have quantitative differences in cholesterol and oxysterol content, but information on the oxysterol profile in fatty streaks is limited. This study aims to provide more detailed oxysterol quantification in human fatty streaks, as well as normal aorta and advanced lesions.Methods: A newly adapted method was used, including oxysterol purification by means of a silica cartridge; and it was ensured that artifactual oxysterol formation was kept to a minimum. Cholesterol and oxysterols were estimated by GC and identification confirmed by GC-MS in samples of normal human arterial intima, intima with near-confluent fatty streaks and advanced lesions, in necropsy samples.Results: The oxysterols 7α-hydroxycholesterol, cholesterol-5β, 6β-epoxide, cholesterol-5α, 6α-epoxide, 7β-hydroxycholesterol, 7-ketocholesterol and 27-hydroxycholesterol (formerly known as 26-hydroxy-cholesterol) were found in all the lesions, but were at most very low in the normal aorta, both when related to wet weight and when related to cholesterol. Most components of the normal artery showed some cross-correlation on linear regression analysis, but cross-correlations were weaker in the fatty streaks and advanced lesions. However, in fatty streak there was a marked positive correlation between 27-hydroxycholesterol and cholesterol.Conclusion: The findings confirm that oxysterols are present in fatty streaks and advanced lesions and may arise from different cholesterol oxidation mechanisms, including free radical-mediated oxidation and enzymatic oxidation.

BACKGROUND:Sex and atherosclerotic plaque histology are intertwined, with fibrous plaques being more prevalent in women. Plaque erosion, a significant contributor to acute coronary syndromes, is linked to fibrous plaques and is more prevalent in women than men. We hypothesize that the molecular drivers of histologically determined fibrous plaques differ between men and women.METHODS:Human end-stage atherosclerotic plaques were isolated from carotid endarterectomy patients included in the Athero-Express Biobank. Fibrous plaques were histologically assessed, linked to clinical characteristics, and processed for protein, bulk RNA, single-cell RNA, and DNA methylation data. We leveraged sex-differential gene expression and deconvolution analyses to uncover sex-biased molecular and cellular mechanisms. Spatial transcriptomics localized gene expression patterns in plaques. Furthermore, we studied the female-biased processes in human plaque endothelial cells and vascular smooth muscle cells stimulated with TGF-β (transforming growth factor-β), with or without SMAD3 (SMAD family member 3) inhibition.RESULTS:Of 1889 atherosclerotic plaques (1309 male and 580 female), fibrous lesions were observed in 50% of female and 31% of male patients. Compared with patients with atheromatous plaques (n=494), women with fibrous plaques exhibited a high prevalence of smoking, while men with fibrous plaques presented more often with diabetes. Female fibrous plaques were characterized by smooth muscle cell–driven ECM (extracellular matrix) remodeling, TGF-β response, and endothelial-to-mesenchymal transition, localized to the fibrous cap. Conversely, male plaques were linked to macrophage-mediated inflammation proximal to the core, dependent on diabetes. Finally, we experimentally confirmed these female-biased mechanisms, showing that TGF-β induced endothelial-to-mesenchymal transition in endothelial cells and ECM remodeling in vascular smooth muscle cells, both partly reversed by SMAD3 inhibition.CONCLUSIONS:Women and men with end-stage fibrous atherosclerotic plaques exhibit distinct clinical and molecular profiles. These mechanisms might be candidate pathways to understand plaque erosion from a molecular point of view and may provide promising targets for atherosclerosis therapies, as they account for both sex and plaque phenotype.

Autophagy in Atherosclerosis: Not All Foam Cells Are Created Equal.

The uptake and incorporation of [I14-C] oleic acid by diseased arterial intima removed by thrombendarteriectomy in 3 patients with Thromboangiitis obliterans (TAO) was studied. The diagnosis of TAO had been established by clinical, angiographic and histological criteria. The uptake and distribution of the label was found very similar in TAO and normal intima and differed considerably from atherosclerotic intima, from fatty streaks as well as from fibro-fatty lesions. In fatty streak lesions the incorporation of [I14-C] oleic acid into phospholipid, triglyceride and cholesterol ester was significantly increased compared to TAO, normal intima and unbilical artery. In TAO the distribution of labeled lipids between subcellular fractions of the arterial intima was also studied and, as in normal intima, most of the cholesterol ester were found membrane-bound whereas in fibro-fatty lesions the bulk of the cholesterol ester was present in the lipid skin fraction. The incorporation of [I14-C] oleic acid into different phospholipids was highest in atherosclerotic intima while no significant differences were found between normal intima and TAO. These data suggest a different pathogenesis of TAO and atherosclerosis.

Fatty acids (e.g. 16:1n-7) and desaturase indices (e.g. stearoyl-CoA desaturase, SCD) in plasma cholesteryl esters (CE) and phospholipids (PL) are used as biomarkers of dietary fat quality and lipid metabolism and are associated with disease outcomes. Endogenously produced circulating fatty acids are believed to reflect composition of the liver, yet little data exist to support such relationship. We investigated associations between circulating fatty acids and fatty acids within the liver. Liver biopsies and blood were collected from n=60 patients with non-alcoholic fatty liver disease. Fatty acids in CE, PL and triglycerides (TG) in plasma and liver were analyzed using gas chromatography. Associations were assessed using Spearman rank correlations. Overall, fatty acids and desaturase indices in plasma PL and TG showed moderate-strong correlations with fatty acids and desaturase indices in corresponding lipid fractions in liver. For plasma CE, 16:1n-7 and SCD were correlated with 16:1n-7 and SCD in liver CE. Noteworthy, fatty acids in plasma CE and PL also showed moderate-strong correlations with fatty acids in liver TG (e.g. r=0.82-0.87 for 16:1n-7 and r=0.77 for SCD). We demonstrate that fatty acids in circulating lipid fractions, including CE, TG and PL, reflects the composition of liver TG in humans, suggesting that circulating fatty acids might be useful biomarkers for the fatty acid composition of the liver. As liver tissue is rarely available in cohort studies, our findings could enhance our understanding of plasma fatty acids as markers of hepatic lipid metabolism and their links to metabolic diseases.

Aortic and coronary atherosclerosis in the Malaysian Orang Asli

Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

1. The influence of chronic treatment with molsidomine, pro-drug of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on fatty streak development and release of NO and prostacyclin (PGI2) was studied in the aorta of normal and cholesterol-fed rabbits. 2. Groups of 10 rabbits received standard diet (150 g day-1), or diets with 0.3% cholesterol, with 0.02% molsidomine or with the combination of cholesterol and molsidomine for 16 weeks. Lesion area and thickness, maximum change in isometric force (Emax) and sensitivity (-log EC50 or pD2) to constricting and relaxing agonists were assessed in segments of arch, thoracic and abdominal aorta. Bioassay was used to assess NO release. 3. Cholesterol-induced fatty streaks tapered off towards the abdominal aorta. Area, thickness, weight and cholesterylester content of the lesions were augmented by the NO donor, whereas the hypercholesterolaemia remained unchanged. The exacerbation was attributed to co-release of superoxide anion from the sydnonimine. 4. As fatty streaks progressed, amplitude and pD2 of acetylcholine (ACh)-induced relaxations decreased, whereas cyclic GMP and cyclic AMP second messenger systems were not influenced, since Emax and sensitivity to SIN-1 and forskolin remained unchanged. However, extensive lesions apparently trapped some NO, as the pD2 of authentic NO decreased. 5. The fatty streaks curtailed the biosynthesis of PGI2 and the overflow of NO from the perfused thoracic aorta. The latter defect was not restored by L-arginine and appears to be consistent with a functional change of the endothelial muscarinic receptors. 6. The NO donor desensitized the aorta to cyclic GMP-mediated relaxations (ACh, SIN-1 and NO), without affecting cyclic AMP-mediated relaxation to forskolin or constrictor responses to phenylephrine and 5-hydroxytryptamine. 7. The drug also suppressed the ACh-induced overflow of NO, without changing PGI2 release. This selective reduction of endothelial NO release and the desensitization of cyclic GMP-mediated relaxations occurred independently of fatty streak formation. 8. The results indicate that chronic exposure to exogenous NO downregulates endothelial NO release and cyclic GMP-mediated relaxations, and provide evidence for the existence of negative feed-back regulations of the L-arginine NO pathway under in vivo conditions.

SummaryIsotopic cholesterol was administered per os for 30 days to cholesterol-fed White Carneau pigeons. At weekly intervals and for 100 days after cessation of isotope administration, subgroups were killed and their aortas were divided into normal tissue, fatty streaks, and atherosclerotic plaques. Cryostat sections from each plaque were examined for morphologic features, and from the different areas of the aorta cholesterol concentration and radioactivity were determined. From the values obtained, rates of influx and efflux of plasma cholesterol were calculated. The results indicated that in normal aorta the influx of free cholesterol greatly exceeds that of cholesteryl esters. There appeared to be no net positive cholesterol balance. In fatty streaks, the influx of both free and esterified cholesterol was greater than for normal tissue, and influx rates exceeded those for efflux. In plaques, still greater rates of influx and efflux were seen, and the influx of cholesteryl esters frequently exceeded th...

Lipoprotein metabolism in FIC1 deficiency due to ATP8B1 mutations has never been studied sufficiently. This study was performed to investigate the detailed lipoprotein metabolism in benign recurrent intrahepatic cholestasis (BRIC) caused by FIC1 deficiency. Lipoprotein profile and major lipoprotein regulators such as lecithin:cholesterol acyltransferase (LCAT), hepatic triglyceride lipase (HTGL), lipoprotein lipase, and cholesteryl ester transfer protein in a Japanese patient with BRIC were serially examined during a bout of cholestasis. Liver expression of farnesoid X receptor (FXR), which suppresses high-density lipoprotein (HDL) generation, was also examined. Hypercholesterolemia and lipoprotein X accumulation were never observed throughout this study. When the cholestasis was severe, triglyceride-rich low-density lipoprotein (LDL) accounted for most of the plasma lipoproteins whereas HDL was hardly detectable. Concurrently, activities of all regulators were decreased, together with decreases of the serum parameter for liver protein synthesis. In particular, suppressions of LCAT and HTGL activities were severe and greatly contributed to the appearance of triglyceride-rich LDL. As the cholestasis improved, this LDL gradually transformed into normal LDL with the recoveries of LCAT and HTGL activities. The activities of all regulators for the last 1 to 2 months were normal but HDL remained depleted. His liver showed low FXR expression compared with control livers. The present study showed an appearance of triglyceride-rich LDL due to suppressions of LCAT and HTGL activities and a depletion of HDL that is not able to be explained by lipoprotein regulators or FXR in our patient.

Studies were carried out for 2.5 to 3 years in adult male African green monkeys (grivet subspecies) fed diets containing 22% of calories as lard or fish oil with 40% of calories as fat and 0.75 mg cholesterol/Kcal to determine if isocaloric substitution of menhaden fish oil for lard affects coronary artery atherosclerosis. The average total plasma cholesterol concentrations during the experimental period were significantly lower for the fish-oil group (231 +/- 37 mg/dl) compared to the lard group (360 +/- 44 mg/dl), but this difference did not become apparent until after 5 months of experimental diet consumption. High density lipoprotein cholesterol concentrations were 30% lower (p less than 0.01) for the fish-oil group also (57 +/- 5 vs. 82 +/- 6 mg/dl). Plasma triglyceride concentrations were low for both groups, but after about 5 months of diet consumption, they were higher for the animals fed fish oil (25 +/- 2 mg/dl) compared to their lard-fed counterparts (15 +/- 1 mg/dl). Coronary artery intimal area (in this case a measure of early atherosclerotic lesion size) was low in all animals but was significantly less (p less than 0.03) for the fish oil vs. lard groups (0.01 +/- 0.002 vs. 0.03 +/- 0.009 mm2). More atherosclerosis was found in other arteries, and a trend was seen of less atherosclerosis in the thoracic aorta and common carotid arteries of the fish-oil group. The size of lesions in the abdominal aorta was similar between diet groups, but microscopic examination of arteries of the lard group revealed relatively more cholesterol monohydrate crystals compared to the arteries of the fish-oil group. Chemical analysis showed that there was less esterified cholesterol (1.46 +/- 0.71 vs. 3.43 +/- 0.74 mg/g, p = 0.04) and free cholesterol (3.7 +/- 2.15 vs. 7.05 +/- 1.68 mg/g, p = 0.08) in the abdominal aortas taken from the animals fed fish oil. There was a significant correlation between low density lipoprotein (LDL) cholesteryl ester (CE) fatty acid ratio (i.e., saturated + monounsaturated/polyunsaturated species) and the amount of esterified (r = 0.59) and free (r = 0.63) cholesterol in the abdominal aortas. Compared to the lard group, animals fed fish oil had significantly lower LDL CE melting temperatures (26 +/- 1 vs. 38 +/- 1 degree C) and significantly smaller LDL particles (2.68 +/- 0.10 vs. 3.25 +/- 0.38 g/mumol). Therefore, the potentially antiatherogenic effects of dietary fish oil include its ability to decrease the concentration, size, CE content, and CE melting temperature of plasma LDL.

This study has investigated in detail factors regulating accumulation, esterification, and mobilization of cholesterol in human THP-1 macrophages. Human THP-1 monocytes were differentiated into macrophages and then cholesterol enriched by exposure to acetylated LDL (AcLDL), together with [3H]free cholesterol (FC). Although THP-1 macrophages accumulated FC and esterified cholesterol (EC), assessed by both mass and radioactivity, cellular EC always demonstrated a much lower specific activity (cpm/ microg) than did cellular FC, and several potential causes of this finding were investigated. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) during loading decreased cell [3H]EC by 95+/-1.4% but decreased cell EC mass by only 66.0+/-4.0%, indicating that some intracellular undegraded AcLDL-derived EC was present in these cells. Esterification of [3H]oleate to EC in THP-1 cells loaded with AcLDL was 2.0 nmol x mg-1 x h-1, consistent with previous literature. However, EC, triglyceride, and phospholipid fractions respectively contained 1.0+/-0.07%, 80.0+/-0.5%, and 18.9+/-0.3% of cell [3H]oleate, indicating triglycerides were much more metabolically active than EC. In addition, the mass of triglyceride in THP-1 macrophages exceeded that of EC both before and after exposure to AcLDL. Esterification of nonlipoprotein-derived cholesterol was compared in THP-1 cells and nonhuman Fu5AH, CHO, and RAW macrophage cells. Whereas the nonhuman cell lines all esterified over 30% of 2-hydroxypropyl-beta-cyclodextrin (hp-ss-CD)-delivered cholesterol within 6 hours, THP-1 cells esterified <8.0% of incorporated cholesterol. Kinetics of cholesterol efflux from AcLDL-loaded THP-1 cells were first investigated after loading with only FC, and interactions between efflux and EC hydrolysis were further assessed after loading cells with both EC and FC. Over 24 hours, human apolipoprotein (apo) A-I, apoHDL reconstituted with phosphatidylcholine, and HDL3 respectively removed 46.6+/-3.7%, 61. 3+/-3.4%, and 76.4+/-10.1% of [3H]FC from FC-enriched THP-1 cells. Cholesterol efflux to apoA-I was saturated by 24 hours and was enhanced by using apoA-I-phospholipid instead of pure apoA-I. Kinetic modeling identified that 97% of effluxed FC derived from a slow pool, with a T1/2 ranging from 27.7 hours for HDL to 69.3 hours for apoA-I. Although efflux enhanced net clearance of EC, hydrolysis of EC during concurrent inhibition of ACAT was unaffected by cholesterol efflux. Supplementation of THP-1 cultures with cAMP to stimulate hormone-sensitive lipase did not significantly enhance net hydrolysis of EC or cholesterol efflux. In conclusion, human THP-1 macrophages contain a large and metabolically active pool of triglyceride and a relatively inactive pool of EC. The low specific activity of EC relative to FC is contributed to by reduced esterification of FC, slow hydrolysis of EC, and accumulated lipoprotein EC. The relative inactivity of the EC pool may further contribute to already impaired cholesterol efflux from these cells. Net cholesterol efflux from human macrophages is achieved by pure apoA-I and is substantially further enhanced by the presence of phospholipid in acceptor particles.

Liquid crystal monomers (LCMs) are new pollutants derived from emissions from liquid crystal displays (LCDs). In recent years, environmental and health concerns on LCMs have mainly focused on nematic and smectic LCMs. However, information regarding cholesteric LCMs in the environment remains lacking. Herein, we made the first comprehensive screening of cholesteric LCMs in e-waste dust. Target analysis uncovered the presence of 28 cholesteric LCMs in dust samples, including 14 fatty acid cholesteryl esters (FACEs), 9 alkyl carbonate cholesteryl esters (ACCEs), and 5 other-type cholesteryl esters. According to the analogous characteristics of FACEs, a suspect screening framework identified an additional 43 long-chain FACEs. Overall, 71 cholesteric LCMs were identified, with 30 detected in >50% of samples. The median and highest values of their sum concentrations were 94.3 and 1880 ng/g, respectively. FACEs emerged as the dominant group, with long-chain FACEs, particularly FACEs (18:0), exhibiting significantly more abundance than short-chain and medium-chain FACEs. In silico predictive modeling revealed that most of these newly identified cholesteric LCMs exhibited medium-to-high estrogenic/androgenic activities. Our findings confirm cholesteric LCMs as another abundant class of LCD-derived new pollutants in the environment and highlight the need to include these new chemicals in the future risk assessment of LCMs.