Inflammasome Activation Aggravates Cutaneous Xanthomatosis and Atherosclerosis in ACAT1 (Acyl-CoA Cholesterol Acyltransferase 1) Deficiency in Bone Marrow.

Objective- ACAT1 (Acyl-CoA cholesterol acyltransferase 1) esterifies cellular free cholesterol, thereby converting macrophages to cholesteryl ester-laden foam cells in atherosclerotic lesions and cutaneous xanthoma. Paradoxically, however, loss of ACAT1 in bone marrow causes the aggravation of atherosclerosis and the development of severe cutaneous xanthoma in hyperlipidemic mice. Recently, it has been reported that cholesterol crystals activate NLRP3 (NACHT, LRR [leucine-rich repeats], and PYD [pyrin domain] domain-containing protein 3) inflammasomes, thereby contributing to the development of atherosclerosis. The present study aimed to clarify the role of NLRP3 inflammasomes in the worsening of atherosclerosis and cutaneous xanthoma induced by ACAT1 deficiency. Approach and Results- Ldlr-null mice were transplanted with bone marrow from WT (wild type) mice and mice lacking ACAT1, NLRP3, or both. After the 4 types of mice were fed high-cholesterol diets, we compared their atherosclerosis and skin lesions. The mice transplanted with Acat1-null bone marrow developed severe cutaneous xanthoma, which was filled with numerous macrophages and cholesterol clefts and had markedly increased expression of inflammatory cytokines, and increased atherosclerosis. Loss of NLRP3 completely reversed the cutaneous xanthoma, whereas it improved the atherosclerosis only partially. Acat1-null peritoneal macrophages showed enhanced expression of CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein) and TNF-α (tumor necrosis factor-α) but no evidence of inflammasome activation, after treatment with acetylated LDL (low-density lipoprotein). Conclusions- Elimination of ACAT1 in bone marrow-derived cells aggravates cutaneous xanthoma and atherosclerosis. The development of cutaneous xanthoma is induced mainly via the NLRP3 inflammasome activation.

Aberrant tyrosinase expression in an atypical fibroxanthoma: A case report.

Atypical fibroxanthoma (AFX) is a histologic mimicker of a variety of spindle cell neoplasms, and careful microscopic and immunohistochemical evaluation is critical in establishing the correct diagnosis. Here we report the histologic and immunohistochemical work up of a 1 cm nodule involving the left dorsal hand of a 66-year-old patient. Light microscopy revealed fascicles of spindled and pleomorphic cells within the dermis showing increased mitotic activity occurring in the background of sun-damaged skin. There were numerous multinucleated cells with hyperchromatic nuclei and ample finely vacuolated or foamy cytoplasms. There was strong and diffuse CD10 and patchy CD68 expression among the spindled cells and multinucleated cells. The neoplastic cells did not show immunoreactivity against S100, p75-NGFR, HMB-45 or a panel of keratinocytic, vascular and smooth muscle markers. Tyrosinase and Melan-A were not expressed within the spindle cell component of this neoplasm; however, there was tyrosinase expression among numerous multinucleated giant cells. Melan-A expression was also observed among rare multinucleated giant cells. Tyrosinase expression has not previously been reported in AFX.

Combined "Infiltrating Astrocytoma/Pleomorphic Xanthoastrocytoma" Harboring IDH1 R132H and BRAF V600E Mutations.

Pleomorphic xanthoastrocytoma (PXA) has rarely been reported in combination with infiltrating glioma, historically interpreted as a "collision tumor." Isocitrate dehydrogenase 1 (IDH1) and BRAF V600E mutations are usually not concurrent. The former is typical of adult infiltrating gliomas, and the latter is identified in a variety of primary central nervous system neoplasms, including PXA, ganglioglioma, pilocytic astrocytoma, and rarely infiltrating gliomas. We report the case of a 56-year-old man presenting with seizures and headaches. Magnetic resonance imaging revealed a large right temporal lobe mass with low T1 and high T2/FLAIR signal and a discrete contrast-enhancing focus. Histologically, the tumor showed 2 distinct components: an infiltrating astrocytoma harboring 5 mitoses/10 high-power fields and a relatively circumscribed focus, resembling PXA with, at most, 2 mitoses/10 high-power fields. No microvascular proliferation or necrosis was present in either component. The infiltrating astrocytoma component contained numerous axons, whereas the PXA-like component had sparse axons, as demonstrated by the neurofilament immunostain. Both components were positive for the mutant IDH1 R132H and showed loss of ATRX expression, whereas BRAF V600E was restricted to the PXA-like component. On sequencing of the 2 components separately after microdissection, both showed identical IDH1 R132H and TP53 R273C point mutations, whereas the BRAF V600E mutation was limited to the PXA-like component. These findings are consistent with clonal expansion of a morphologically distinct focus, harboring a private BRAF V600E mutation within an IDH1-mutant glioma. Intratumoral heterogeneity and clonal evolution, as seems to have occurred here, suggest reevaluation of "collision tumors" as a concept.

TERT promoter mutations are frequent in atypical fibroxanthomas and pleomorphic dermal sarcomas.

Activating mutations in the TERT promoter leading to increased telomerase expression were recently identified in cutaneous melanoma and subsequently in many other types of cancer. These mutations lead to increased telomerase expression, allowing cells to proliferate continuously without entering apoptosis or senescence. Atypical fibroxanthomas and pleomorphic dermal sarcomas are genetically poorly understood tumors developing in the skin of older patients. Known genetic events in these tumors are mutations in TP53 (atypical fibroxanthoma and pleomorphic dermal sarcoma) and RAS (pleomorphic dermal sarcoma) genes, often having a UV signature. We analyzed a cohort of 27 atypical fibroxanthomas and 34 pleomorphic dermal sarcomas for the presence of TERT promoter mutations by conventional Sanger sequencing. TERT promoter mutations were identified in 25 (93%) atypical fibroxanthomas and in 26 (76%) pleomorphic dermal sarcomas. Mutations were found to have a UV signature (C>T or CC>TT) and were largely identical to those detected in cutaneous melanoma. Our data show that TERT promoter mutations are the most frequent mutations in atypical fibroxanthomas and pleomorphic dermal sarcomas reported to date. The identified mutations confirm the pathogenetic role of UV exposure in both atypical fibroxanthomas and pleomorphic dermal sarcomas and suggest that telomere maintenance through increased expression of telomerase plays an important role in the pathogenesis of these tumors.

A case of hepatitis C-associated osteosclerosis with xanthogranulomatous cholecystitis.

A 62-year-old woman presented with a markedly increased serum ALP level of skeletal origin during a regular follow-up of chronic hepatitis C. Serum calcium, phosphorus, and intact-PTH levels were normal and bone turnover markers were increased. Her generalized bone density was diffusely increased. These findings were consistent with hepatitis C-associated osteosclerosis (HCAO). She underwent cholecystectomy, as gallbladder cancer was suspected; however, histopathological findings demonstrated xanthogranulomatous cholecystitis. After cholecystectomy, serum ALP level and bone turnover markers were gradually decreased. This may indicate the existence of a novel osteogenic factor in the gallbladder in HCAO.

A case of adolescent hyperlipoproteinemia with xanthoma and acute pancreatitis, associated with decreased activities of lipoprotein lipase and hepatic triglyceride lipase.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) enhance the hydrolysis of triglycerides (TG) transported by chylomicron (CM) and very-low-density lipoprotein (VLDL). We report a case of severe hyperchylomicronemia with high levels of remnant lipoprotein and total cholesterol (T-Chol) in a 15-year-old boy. Precise examination of the lipid profile showed decreased activities of both LPL and HTGL, although the protein mass for LPL and HTGL were maintained. In addition, bezafibrate treatment effectively ameliorated hypertriglyceridemia in this case. This is the first case of hyperchylomicronemia with decreased activities and unaffected protein masses for both LPL and HTGL, without overt immuno-dysfunction.

Xanthoma of bone associated with lipoprotein lipase deficiency.

Lipoprotein lipase (LPL) deficiency is an extremely rare congenital metabolic disorder with an accumulation of chylomicrons in the blood. We encountered a patient with an LPL deficiency leading to multiple bone xanthomas associated with hyperlipidemia. Radiographs and MRI of the humerus and femur revealed symmetrical bone lesions, and there is a possibility that these symmetrical lesions may therefore be a characteristic feature for this disorder.

Lapaquistat acetate, a squalene synthase inhibitor, changes macrophage/lipid-rich coronary plaques of hypercholesterolaemic rabbits into fibrous lesions.

BACKGROUND AND PURPOSE: Inhibition of squalene synthesis could transform unstable, macrophage/lipid-rich coronary plaques into stable, fibromuscular plaques. We have here treated WHHLMI rabbits, a model for coronary atherosclerosis and myocardial infarction, with a novel squalene synthase inhibitor, lapaquistat acetate (TAK-475). EXPERIMENTAL APPROACH: Young male WHHLMI rabbits were fed a diet supplemented with lapaquistat acetate (100 or 200 mg per kg body weight per day) for 32 weeks. Serum lipid levels were monitored every 4 weeks. After the treatment, lipoprotein lipid and coenzyme Q10 levels were assayed, and coronary atherosclerosis and xanthomas were examined histopathologically or immunohistochemically. From histopathological and immunohistochemical sections, the composition of the plaque was analysed quantitatively with computer-assisted image analysis. Xanthoma was evaluated grossly. KEY RESULTS: Lapaquistat acetate decreased plasma cholesterol and triglyceride levels, by lowering lipoproteins containing apoB100. Development of atherosclerosis and xanthomatosis was suppressed. Accumulation of oxidized lipoproteins, macrophages and extracellular lipid was decreased in coronary plaques of treated animals. Treatment with lapaquistat acetate increased collagen concentration and transformed coronary plaques into fibromuscular plaques. Lapaquistat acetate also suppressed the expression of matrix metalloproteinase-1 and plasminogen activator inhibitor-1 in the plaque and increased peripheral coenzyme Q10 levels. Increased coenzyme Q10 levels and decreased very low-density lipoprotein cholesterol levels were correlated with improvement of coronary plaque composition. CONCLUSION AND IMPLICATIONS: Inhibition of squalene synthase by lapaquistat acetate delayed progression of coronary atherosclerosis and changed coronary atheromatous plaques from unstable, macrophage/lipid accumulation-rich, lesions to stable fibromuscular lesions.

Cyclooxygenase expression in the gallbladder.

The COX expressions were evaluated separately in the epithelium and in the stroma of gallbladder cancer, chronic cholecystitis, xanthogranulomatous cholecystitis (XGC) and the normal gallbladder. In normal gallbladder COX-2 expression rate was significantly higher in the epithelium than in the stroma. The COX-2 expression rate in the epithelium of non-cancerous adjacent epithelium to cancerous lesion was significantly lower than those not only of cancer, but also chronic cholecystitis, XGC and normal gallbladder. In stroma, the COX-2 expression rate in cancer, chronic cholecystitis and XGC were significantly higher than that of the normal gallbladder. The rate in non-cancerous adjacent stroma to cancer is significantly lower than that of cancer and XGC. However, the difference of rate between of normal and of chronic cholecystitis was not significant. The COX-2 expression rates were significantly higher in both the epithelium and the stroma in the well and moderately differentiated cancer group than in the poorly and undifferentiated cancer group. Our results suggest that COX-2 expression in the gallbladder may be regulated by various factors and not directly related to carcinogenesis. The significance of its repression in the non-cancerous adjacent tissue to cancer lesion should be re-evaluated.

Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia.

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes esterification of cellular cholesterol. To investigate the role of ACAT-1 in atherosclerosis, we have generated ACAT-1 null (ACAT-1-/-) mice. ACAT activities were present in the liver and intestine but were completely absent in adrenal, testes, ovaries, and peritoneal macrophages in our ACAT-1-/- mice. The ACAT-1-/- mice had decreased openings of the eyes because of atrophy of the meibomian glands, a modified form of sebaceous glands normally expressing high ACAT activities. This phenotype is similar to dry eye syndrome in humans. To determine the role of ACAT-1 in atherogenesis, we crossed the ACAT-1-/- mice with mice lacking apolipoprotein (apo) E or the low density lipoprotein receptor (LDLR), hyperlipidemic models susceptible to atherosclerosis. High fat feeding resulted in extensive cutaneous xanthomatosis with loss of hair in both ACAT-1-/-:apo E-/- and ACAT-1-/-:LDLR-/- mice. Free cholesterol content was significantly increased in their skin. Aortic fatty streak lesion size as well as cholesteryl ester content were moderately reduced in both double mutant mice compared with their respective controls. These results indicate that the local inhibition of ACAT activity in tissue macrophages is protective against cholesteryl ester accumulation but causes cutaneous xanthomatosis in mice that lack apo E or LDLR.

Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA:cholesterol acyltransferase 1.

Inhibitors of acyl CoA:cholesterol acyltransferase (ACAT) have attracted considerable interest as a potential treatment for atherosclerosis. Currently available inhibitors probably act nonselectively against the two known ACATs. One of these enzymes, ACAT1, is highly expressed in macrophages in atherosclerotic lesions, where it contributes to foam-cell formation. In this study, we examined the effects of selective ACAT1 deficiency in two mouse models of atherosclerosis. In the setting of severe hypercholesterolemia caused by deficiency in apoE or the LDL receptor (LDLR), total ACAT1 deficiency led to marked alterations in cholesterol homeostasis and extensive deposition of unesterified cholesterol in the skin and brain. Bone marrow transplantation experiments demonstrated that ACAT1 deficiency in macrophages was sufficient to cause dermal xanthomas in hyperlipidemic LDLR-deficient mice. ACAT1 deficiency did not prevent the development of atherosclerotic lesions in either apoE-deficient or LDLR-deficient mice, despite causing relatively lower serum cholesterol levels. However, the lesions in ACAT1-deficient mice were atypical in composition, with reduced amounts of neutral lipids and a paucity of macrophages in advanced lesions. Although the latter findings may be associated with increased lesion stability, the marked alterations in cholesterol homeostasis indicate that selectively inhibiting ACAT1 in the setting of severe hyperlipidemia may have detrimental consequences.

HMG-CoA reductase is not the site of the primary defect in phytosterolemia.

Phytosterolemia is an autosomal recessive disorder characterized by the excessive absorption, reduced excretion, and consequent high tissue and plasma levels of plant sterols, by the presence of tendon xanthomas, and by premature atherosclerosis. Low HMG-CoA reductase (HRase) activity and mass have been reported in liver and mononuclear leucocytes and low mRNA levels in liver from phytosterolemic subjects. These results led to the proposal that the primary defect in this condition involves the HRase gene locus. We examined this hypothesis in phytosterolemic subjects and heterozygous parents from four unrelated families. A variable number tandem repeat (VNTR) polymorphism of the HRase gene in the three informative families and a ScrFI restriction fragment length polymorphism (RFLP) within intron 2 of the gene in one of these families, segregated independently of the disease phenotype. Biological parentage was confirmed in the family in whom both polymorphisms failed to segregate with the disorder. These results conclusively exclude the HRase gene locus as the site of the primary defect in phytosterolemia. The study was extended by examining plasma levels of mevalonic acid and lathosterol, both markers of cholesterol biosynthesis, in response to cholestyramine, a bile acid sequestrant that is known to up-regulate HRase. Oral administration of cholestyramine resulted in a substantial (7.7-fold) increase in mevaIonic acid levels in two phytosterolemic subjects, compared with a 2.2-fold rise in their obligate heterozygote parents and a 2.3-fold increase in three healthy control subjects. The lathosterol/cholesterol (L/C) ratio showed a quantitatively similar response. Baseline levels of mevalonate and the L/C ratio were low in the phytosterolemic patients in conformity with reports of reduced cholesterol biosynthesis and HRase activity in this disorder. These functional data provide support for the concept that the primary defect in phytosterolemia does not affect a trans gene locus responsible for the constitutive expression or regulation of HMG-CoA reductase.

Japanese triplets with cerebrotendinous xanthomatosis are homozygous for a mutant gene coding for the sterol 27-hydroxylase (Arg441Trp).

We present the first case of triplets with cerebrotendinous xanthomatosis (CTX). A C-to-T base change identified in the genomic DNA and cDNA encoding the sterol 27-hydroxylase led to replacement of arginine by tryptophan at position 441 (Arg441Trp) in the triplets. The triplets were homozygous and their mother was heterozygous for this mutant gene. The triplets exhibited an identical phenotypic expression, which was different from that of a sporadic CTX case with the same mutation.

Mitochondrial enzyme deficiency in cerebrotendinous xanthomatosis.

We report reduced respiratory chain enzyme activity in isolated muscle mitochondria and high levels of serum and CSF pyruvate and lactate in a case of cerebrotendinous xanthomatosis. These findings raise the question of the pathogenetic role of mitochondrial dysfunction in this syndrome.

Identification of new mutations in sterol 27-hydroxylase gene in Japanese patients with cerebrotendinous xanthomatosis (CTX).

Cerebrotendinous xanthomatosis (CTX) is a hereditary sterol storage disease associated with accumulation of cholesterol and cholestanol in various tissues, especially tendons and neural tissues. The biochemical defect that causes CTX is a deficiency of the mitochondrial sterol 27-hydroxylase which oxidizes the side chain of cholesterol in connection with formation of bile acids. Japan has a relatively high prevalence of CTX and more cases of the disease are found here than in any other country. In the present study two new different point mutations are described in the heme-ligand binding domain of the sterol 27-hydroxylase gene in three Japanese CTX patients and one CTX heterozygote. Two of the homozygotes as well as the heterozygote subject have a single base substitution of A for G at codon 441 [CGG (Arg) to CAG (Gln)]. Another homozygote has a transition of C to T at codon 441 [CGG (Arg) to TGG (Trp)]. These two different mutations result in two restriction fragment length polymorphisms (RFLPs) for the enzymes StuI or HpaII. We also assayed sterol 27-hydroxylase activity using skin fibroblasts derived from three CTX patients, one CTX heterozygote, and normal subjects. While two of the homozygous subjects have undetectable levels of the enzyme activity, one homozygous subject and one heterozygous subject have decreased levels of the enzyme activity, about 1.4% and 10% of normal, respectively. The results suggest that the newly identified point mutations in the sterol 27-hydroxylase gene could account for the sterol 27-hydroxylase deficiency in the Japanese CTX patients.

A point mutation in the bile acid biosynthetic enzyme sterol 27-hydroxylase in a family with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare familial disorder characterized by progressive neurological dysfunction, atherosclerosis, and xanthomas with sterol storage in the nervous system, vessels, and tendons. Increased serum cholestanol, derived from intermediates of cholesterol catabolism, may possibly be a major cause of the disease. An examination was made of the cDNA encoding cytochrome P450 sterol 27-hydroxylase (CYP27) in hepatic mitochondria, considered a defective enzyme inducing CTX, in a Japanese housewife afflicted with CTX and her family. The proposita and one of her brothers, who also had CTX symptoms and hypercholestanolemia, were found to be homozygotic, carrying a point mutation in the CYP27 gene at Arg104 (CGG) to Trp104 (TGG). The mutant position has a 100% conserved positive charge in all known vertebrate cytochrome P450s and even in bacterial cytochrome P450cam. The mother of the proposita and another brother were both free of CTX symptoms and were heterozygotic for the mutation, although their plasma cholesterol increased moderately. An increase in plasma cholestanol alone would, thus, not appear to be a direct cause of sterol storage in CTX, while CTX is strongly suggested to be caused by defects in both alleles of the CYP27 gene.

Decreased cholesterol biosynthesis in sitosterolemia with xanthomatosis: diminished mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and enzyme protein associated with increased low-density lipoprotein receptor function.

We investigated the mechanism for reduced cholesterol biosynthesis in sitosterolemia with xanthomatosis. The conversion of acetate to cholesterol and total and active hydroxymethylglutaryl (HMG) coenzyme A (CoA) reductase activities, enzyme protein mass, and catalytic efficiency were related to low-density lipoprotein (LDL) receptor function in freshly isolated mononuclear leukocytes collected at 9 AM after a 12-hour fast from two affected sisters and 12 control subjects. Active HMG-CoA reductase activity was determined in mononuclear leukocyte microsomes prepared and assayed in the presence of sodium fluoride, while total HMG-CoA reductase activity was determined in the absence of the phosphatase inhibitor. Enzyme protein was assayed using rabbit polyclonal anti-rat liver microsomal HMG-CoA reductase serum. The rates at which [14C]acetate was transformed to cholesterol by sitosterolemic mononuclear leukocytes were decreased 29% and 41%, respectively, compared with the mean value for mononuclear leukocytes from 12 control subjects. Similarly, total HMG-CoA reductase activities were 71% and 68% lower in sitosterolemic mononuclear leukocyte microsomes and were associated with 62% and 65% less enzyme protein than the mean for the control microsomal preparations. This marked decrease in HMG-CoA reductase protein mass in sitosterolemic microsomes was partially compensated for by an increase in the proportion of active enzyme. Sitosterolemic plasma and mononuclear leukocyte cholesterol concentrations were not significantly different from control values, although total sterol levels were increased about 20% because of abundant plant sterols. In contrast, receptor-mediated LDL degradation by sitosterolemic mononuclear leukocytes was increased 50% over control.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive inhibition of bile acid synthesis by endogenous cholestanol and sitosterol in sitosterolemia with xanthomatosis. Effect on cholesterol 7 alpha-hydroxylase.

The 7 alpha-hydroxylation of two cholesterol analogues, sitosterol and cholestanol, and their effect on the 7 alpha-hydroxylation of cholesterol were measured in rat and human hepatic microsomes. In untreated rat liver microsomes, the 7 alpha-hydroxylation of cholesterol was higher than that of cholestanol (1.4-fold) and sitosterol (30-fold). After removal of endogenous sterols from the microsomes by acetone treatment, the 7 alpha-hydroxylation of cholesterol was similar to that of cholestanol and only fourfold higher than that of sitosterol. Cholestanol and sitosterol competitively inhibited cholesterol 7 alpha-hydroxylase in both rat and human liver microsomes, with cholestanol the more potent inhibitor. Patients with sitosterolemia with xanthomatosis, who have elevated microsomal cholestanol and sitosterol, showed reduced cholesterol 7 alpha-hydroxylase activity relative to the activity in control subjects (13.9 and 14.7 vs. 20.3 +/- 0.9 pmol/nmol P-450 per min, P less than 0.01). Enzyme activity in these patients was 40% higher when measured in microsomes from which competing sterols had been removed. Ileal bypass surgery in one sitosterolemic patient decreased plasma cholestanol and sitosterol concentrations and resulted in a 30% increase in hepatic microsomal cholesterol 7 alpha-hydroxylase activity. Cholesterol 7 alpha-hydroxylase appears to have a specific apolar binding site for the side chain of cholesterol and is affected by the presence of cholestanol and sitosterol in the microsomal substrate pool. Reduced bile acid synthesis in sitosterolemia with xanthomatosis may be related to the inhibition of cholesterol 7 alpha-hydroxylase activity by endogenous cholesterol analogues.