Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses.

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry.Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathologic intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell-dominant region, and the plasma-derived components (apolipoprotein B and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non-oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes.In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathologic intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

Oxygen levels during negative pressure wound therapy.

AIM OF THE STUDY: Negative pressure wound therapy (NPWT) has become an established treatment modality when dealing with chronic and infected wounds. The underlying mechanism of action is still under discussion and remains controversial. Evidence exists showing rather hypoxic conditions as the main reason for the positive results and bacterial clearance. In an attempt to further explain the mechanism of action, we investigated oxygen levels within the foam interface of a NPWT device. MATERIALS AND METHODS: We used an optical sensor based on the principle of dynamic fluorescence quenching and tested five different commonly available NPWT systems used during our daily clinical routine. All measurements were done in an in vitro experimental design for at least 24 h and multiple vacuum intensities were investigated. RESULTS: Oxygen levels decreased as much as 22.8% and the amount of vacuum applied inversely correlated with the oxygen reduction. A stepwise increase in vacuum of 25 mmHg showed a linear mean drop of 2.75% per setting. All devices were able to maintain a constant level of negative pressure, and no significant difference between the various dressings was found (p > 0.05). CONCLUSION: Therefore, oxygen levels are decreased within the foam of NPWT dressings, likely leading to oxygen deprivation effects in the underlying wound tissue.

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PURPOSE: Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. METHODS: Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. RESULTS: Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. CONCLUSIONS: A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Monitoring lipid peroxidation within foam cells by lysosome-targetable and ratiometric probe.

Lipid peroxidation (LPO) in lysosomes is a valuable analyte because it is close associated with the evolutions of some major diseases. As a typical example, in the start-up phase of atherosclerosis, lysosomes get as swollen as foams, by accumulating a large amount of lipoproteins, which facilitates the free-radical chain propagation of LPO. Despite the existences of several fluorescent LPO probes, they are not appropriate for reporting the local extents of lysosomal LPO, for their unspecific intracellular localizations. Here, Foam-LPO, the first fluorescent LPO probe specifically targeting lysosomes, has been developed through straightforward synthesis using low-cost reagents. A basic tertiary amine group enables it to selectively localize in acidic lysosomes; and the conjugated diene moiety within the BODIPY fluorophore will degrade in response to lipid peroxidation, which results in fluorescence maximum shifting from 586 to 512 nm. Thus, under a confocal fluorescence microscope, Foam-LPO is able not only to visualize dynamic morphological changes of lysosomes during the evolution of foam cells, but also to relatively quantify local LPO extents in single lysosomes through ratiometric imaging. In addition, Foam-LPO proves applicable for two-color flow cytometry (FCM) analysis to make quantitative and high-throughput evaluation of LPO levels in large quantity of cells at different stages during the induction to form foam cells. Also importantly, with the aid of this new probe, the different roles played by low-density lipoprotein (LDL) and its oxidized form (ox-LDL) for the LPO processes of foam cells are distinguished and clarified, which benefits the understanding in the initiation and control factors of atherosclerosis.

Low-density lipoprotein apheresis for proteinuria in lupus nephritis with intraglomerular foam cells containing cholesterol crystals.

A 28-year-old woman with systemic lupus erythematosus was referred to our hospital due to nephrotic-level proteinuria despite approximately 1 year of treatment with 50 to 60 mg/d of prednisolone and 100 to 150 mg/d of cyclosporine with methylprednisolone pulse therapy. Kidney biopsy showed diffuse global lupus nephritis (World Health Organization class 4-G A/C) with many intraglomerular foam cells containing cholesterol crystals. Surprisingly, proteinuria diminished after only 5 low-density lipoprotein (LDL) cholesterol apheresis sessions. This case demonstrated the potential of LDL apheresis to exhibit a remarkable effect on not only focal segmental glomerulosclerosis, but also other types of nephritis, particularly nephritis with intraglomerular foam cells.

Chemerin and CMKLR1 expression in human arteries and periadventitial fat: a possible role for local chemerin in atherosclerosis?

BACKGROUND: Depending on their anatomical location, different fat depots have a different capacity to produce bioactive peptides, called adipokines. Adipokines produced by periadventitial fat have been implicated in the pathogenesis of vascular disease, including atherosclerosis. Chemerin is an adipokine with an established role in immunity, adipose tissue function and metabolism, acting in autocrine, paracrine and endocrine manners. We investigated the protein expression of chemerin and its receptor, CMKLR1, in human aortas, coronary vessels and the respective periadventitial adipose tissue and correlated their expression with the presence of atherosclerosis. METHODS: Immunohistochemistry for chemerin and CMKLR1 was performed on human aortic and coronary artery samples including the periadventitial adipose tissue. Aortic and coronary atherosclerotic lesions were assessed using the AHA classification. RESULTS: Chemerin immunopositivity was noticed in both periadventitial fat depots, in vascular smooth muscle cells and foam cells in atherosclerotic lesions. Periadventitial fat and foam cell chemerin immunopositivity was statistically significantly correlated with the severity of atherosclerosis in both locations. CMKLR1 was expressed in vascular smooth muscle cells and foam cells in aortic and coronary vessels with atherosclerotic lesions. CMKLR1 immunostaining in foam cells was statistically significantly correlated with aortic atherosclerosis. CONCLUSIONS: Our results lend some support to a presumable role of locally produced chemerin in the progression of atherosclerotic lesions, possibly acting through its CMKLR1 receptor. Further research will elucidate the role of chemerin signaling in atherosclerosis.

Networklike propagation of cell-level stress in sheared random foams.

Quasistatic simple shearing flow of random monodisperse soap froth is investigated by analyzing surface evolver simulations of spatially periodic foams. Elastic-plastic behavior is caused by irreversible topological rearrangements (T1s) that occur when Plateau's laws are violated; the first T1 determines the elastic limit and frequent T1 avalanches sustain the yield-stress plateau at large strains. The stress and shape anisotropy of individual cells is quantified by Q, a scalar derived from an interface tensor that gauges the cell's contribution to the global stress. During each T1 avalanche, the connected set of cells with decreasing Q, called the stress release domain, is networklike and nonlocal. Geometrically, the networklike nature of the stress release domains is corroborated through morphological analysis using the Euler characteristic. The stress release domain is distinctly different from the set of cells that change topology during a T1 avalanche. Our results highlight the connection between the unique rheological behavior of foams and the complex large-scale cooperative rearrangements of foam cells that accompany distinctly local topological transitions.

Label-Free Tomographic Imaging of Lipid Droplets in Foam Cells for Machine-Learning-Assisted Therapeutic Evaluation of Targeted Nanodrugs.

Lipid droplet (LD) accumulation, a key feature of foam cells, constitutes an attractive target for therapeutic intervention in atherosclerosis. However, despite advances in cellular imaging techniques, current noninvasive and quantitative methods have limited application in living foam cells. Here, using optical diffraction tomography (ODT), we performed quantitative morphological and biophysical analysis of living foam cells in a label-free manner. We identified LDs in foam cells by verifying the specific refractive index using correlative imaging comprising ODT integrated with three-dimensional fluorescence imaging. Through time-lapse monitoring of three-dimensional dynamics of label-free living foam cells, we precisely and quantitatively evaluated the therapeutic effects of a nanodrug (mannose-polyethylene glycol-glycol chitosan-fluorescein isothiocyanate-lobeglitazone; MMR-Lobe) designed to affect the targeted delivery of lobeglitazone to foam cells based on high mannose receptor specificity. Furthermore, by exploiting machine-learning-based image analysis, we further demonstrated therapeutic evaluation at the single-cell level. These findings suggest that refractive index measurement is a promising tool to explore new drugs against LD-related metabolic diseases.

Giant cells lepromatous leprosy. Diffuse dermatitis with exuberant foreign body giant cells in treated lepromatous leprosy / Células gigantes en lepra lepromatosa. Dermatitis difusa con células gigantes tipo cuerpo extraño exuberantes en lepra lepromatosa tratada

Patients with lepromatous leprosy that have received treatment for many years usually get follow up biopsies for persistent skin lesions or positive bacilloscopy even if the values are lower than in the initial bacilloscopy. We report the case of a 48-year old woman with long-standing lepromatous leprosy of 15 years of evolution, with a bacterial index of 4 in the direct smear and the initial skin biopsy. The patient was treated with multidrug therapy for 32 months although the treatment recommended by the World Health Organization (WHO) is only for 12 months. A skin biopsy was taken to determine if there was an active disease. We observed a diffuse dermal inflammation with numerous foreign body giant cells and vacuolated macrophages (Virchow´s cells). These cells contained granular acid-fast material that was also positive with immunohistochemistry for BCG. There were fragmented bacilli and the BI was 2. These cells were also strongly positive for CD68. The biopsy was interpreted as a residual form of lepromatous leprosy that did not require further multidrug therapy. We have observed similar histological profiles in several cases. The lack of clinical data makes it a histological challenge. The accumulation of lipids in these giant cells is due to bacillary destruction and fusion of vacuolated macrophages. We discuss here the role of bacillary and host lipids in the pathogenesis of lepromatous leprosy. We concluded that there was no need to extend the 12-month multidrug therapy recommended by WHO.

Los pacientes con lepra lepromatosa (LL) que han recibido tratamiento durante años, usualmente tienen seguimiento con biopsias de piel para lesiones persistentes o con baciloscopia positiva, con valores menores a los iniciales. Presentamos una mujer de 48 años con LL de 15 años de evolución, con índice bacilar (IB) 4 en el extendido directo y en la biopsia, que recibió terapia multidroga durante 32 meses, aunque el tratamiento recomendado por la Organización mundial de la salud (OMS) es de 12 meses. Se tomó una biopsia de piel para determinar si la enfermedad estaba activa. Se observó inflamación dérmica difusa con numerosas células gigantes tipo cuerpo extraño y macrófagos vacuolados (células de Virchow). Estas células, CD68 positivas, contenían material granular ácido-alcohol resistente, positivo con inmunohistoquímica para BCG. Se encontraron bacilos fragmentados y el IB fue de 2. Se interpretó como una forma residual de LL y que la paciente no requería MDT adicional. Este perfil histológico lo hemos observado en casos similares. Sin datos clínicos estas biopsias son un reto diagnóstico. La acumulación de lípidos en estas células gigantes se debe a la destrucción bacilar y a la fusión de macrófagos vacuolados. Revisamos el papel de los lípidos del bacilo y del huésped en la patogénesis de la LL. En estos casos no es necesario extender los 12 meses de MDT recomendados por la OMS. En el seguimiento de los pacientes se recomienda contar con los hallazgos clínicos, la baciloscopia, la biopsia anual de piel y los títulos IgM anti-glicolípido fenólico.

[Trimetazidine reduces cholesterol content of foam cells by up-regulating autophagy and macrophage extracellular traps, and inhibiting inflammasome].

Objective To explore the effects of trimetazidine (TMZ) on the foam cell formation and its underlying mechanism in oxidized low-density lipoprotein (ox-LDL)-induced macrophages. Methods RAW264.7 cells were stimulated with ox-LDL to establish a foam cell model derived from macrophages. In the experiment, the foam cells were divided into control group (medium), model group (ox-LDL 60 µg/mL), chloroquine (CQ) group (ox-LDL 60 µg/mL combined with CQ 10 mmol/L) and TMZ group (ox-LDL 60 µg/mL combined with TMZ 80 µmol/L, CQ 10 mmol/L). The formation of foam cells was measured by oil red O staining. The levels of total cholesterol (TC) and free cholesterol (FC) in the foam cells were detected using ELISA. Autophagy was observed using MDC staining. Picogreen staining and quantitative fluorescence were performed to detect the formation of macrophage extracellular traps (MET). Then, the mRNA expression of LC3B, P62, inflammasome (NLRP3, ASC, caspase-1) and cholesterol efflux-related genes (ABCA1, ABCG1) were examined by real-time quantitative PCR. Results Compared with the model group, the expression of LC3B mRNA increased significantly, and P62 expression decreased in TMZ combined with CQ group. Meanwhile, the levels of TC, FC, CE/TC, and the expression of NLRP3, ASC, caspase-1 in TMZ combined with CQ group were reduced obviously. Furthermore, the formation of MET in TMZ combined with CQ group was promoted. However, the CQ group showed the opposite results. Conclusion Trimetazidine promotes cholesterol efflux in foam cells via activating autophagy and increasing the formation of MET, as well as inhibiting NLRP3 inflammasome.

α- and ß-d-Glucans from the edible mushroom Pleurotus albidus differentially regulate lipid-induced inflammation and foam cell formation in human macrophage-like THP-1 cells.

Macrophages play an essential role in lipid metabolism; however, the excessive uptake of modified lipids and cholesterol crystals (CC) leads to the formation of pro-inflammatory lipid-laden macrophages called foam cells. Since the α-1,6- and ß-1,3-d-glucans from the basidiome and the mycelium of the edible mushroom Pleurotus albidus have previously been shown to regulate macrophage function, these glucans were tested in macrophage-like THP-1 cells previously exposed to acetylated low-density lipoproteins (acLDL) or CC. The glucans inhibited lipid-induced inflammation, but only the ß-1,3-d-glucan regulated both the NLRP3 inflammasome activation and the expression of genes involved on lipid efflux in acLDL- or CC-pretreated cells, thereby reducing foam cell formation. In contrast, the two α-1,6-glucans tested inhibited foam cell formation only in acLDL-pretreated cells and had no effect on the expression of the peroxisome proliferator-activated receptor gamma and liver X receptor alpha genes, suggesting that these glucans regulate lipid influx rather than lipid efflux. Thus, α- and ß-d-glucans differentially regulate lipid-induced inflammation and foam cell formation in macrophage-like cells. Furthermore, results emphasize that P. albidus has potential to be used as a functional food or as a source for the extraction of biologically-active glucans.

Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol.

Localization of distinct F2-isoprostanes in human atherosclerotic lesions.

F2-Isoprostanes are prostaglandin (PG) isomers formed in situ in cell membranes by peroxidation of arachidonic acid. 8-epi PGF2alpha and IPF2alpha-I are F2-isoprostanes produced in humans which circulate in plasma and are excreted in urine. Measurement of F2-isoprostanes may offer a sensitive, specific, and noninvasive method for measuring oxidant stress in clinical settings where reactive oxygen species are putatively involved. We determined whether isoprostanes were present in human atherosclerotic lesions, where lipid peroxidation is thought to occur in vivo. 8-epi PGF2alpha ranged from 1.310-3.450 pmol/micromol phospholipid in atherectomy specimens compared with 0.045-0.115 pmol/micromol phospholipid (P < 0.001) in vascular tissue devoid of atherosclerosis. Corresponding values of IPF2alpha-I were 5.6-13.8 vs. 0.16-0.44 pmol/micromol phospholipid (P < 0.001). Levels of the two isoprostanes in vascular tissue were highly correlated (r = 0.80, P < 0.0001). Immunohistochemical studies confirmed that foam cells adjacent to the lipid necrotic core of the plaque were markedly positive for 8-epi PGF2alpha. These cells were also reactive with anti-CD68, an epitope specific for human monocyte/macrophages. 8-epi PGF2alpha immunoreactivity was also detected in cells positive for anti-alpha-smooth muscle actin antibody, which specifically recognizes vascular smooth muscle cells. Our results indicate that 8-epi PGF2alpha and IPF2alpha-I, two distinct F2-isoprostanes and markers of oxidative stress in vivo, are present in human atherosclerotic plaque. Quantitation of these chemically stable products of lipid peroxidation in target tissues, as well as in biological fluids, may aid in the rational development of antioxidant drugs in humans.

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo.

Expression of lipoprotein lipase (LPL) by the macrophage has been proposed to promote foam cell formation and atherosclerosis, primarily on the basis of in vitro studies. LPL-deficient mice might provide a model for testing the role of LPL secretion by the macrophage in an in vivo system. Unfortunately, homozygous deficiency of LPL in the mouse is lethal shortly after birth. Because the fetal liver is the major site of hematopoiesis in the developing fetus, transplantation of C57BL/6 mice with LPL-/- fetal liver cells (FLCs) was used to investigate the physiologic role of macrophage LPL expression in vivo. Thirty-four female C57BL/6 mice were lethally irradiated and reconstituted with FLCs from day 14 LPL+/+, LPL+/-, and LPL-/- donors. No significant differences were detected in plasma levels of post-heparin LPL activity or in serum cholesterol or triglyceride levels between the 3 groups on either a chow diet or an atherogenic diet. After 19 weeks on the atherogenic diet, aortae were collected for quantitative analysis of the extent of aortic atherosclerosis. LPL expression was detected by immunocytochemistry and in situ hybridization in macrophages of aortic atherosclerotic lesions of LPL+/+-->C57BL/6 and LPL+/--->C57BL/6 mice, but not in LPL-/--->C57BL/6 mice, whereas myocardial cells expressed LPL in all groups. The mean aortic lesion area was reduced by 55% in LPL-/--->C57BL/6 mice compared with LPL+/+-->C57BL/6 mice and by 45% compared with LPL+/--->C57BL/6 mice, respectively. These data demonstrate in vivo that LPL expression by macrophages in the artery wall promotes foam cell formation and atherosclerosis. off

Role of redox factor-1 in hyperhomocysteinemia-accelerated atherosclerosis.

Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. We have previously shown that homocysteine can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes in vitro. In the present study, we investigated whether redox factor-1 (Ref-1) is involved in HHcy-accelerated atherosclerosis. We used a mild HHcy animal model, aortic roots and peritoneal macrophages were isolated for immunohistochemistry and Western blotting, from apoE-/- and C57BL/6J mice fed a high Hcy diet (1.8 g/L) for 4 or 12 weeks. Four-week HHcy apoE-/- mice showed more plaques and significantly increased immunostaining of Ref-1 and MCP-1 in foam cells, and HHcy mice showed enhanced Ref-1 expression in peritoneal macrophages. To explore the mediating mechanism, incubation with Hcy (100 microM) increased Ref-1 protein level and translocation in human monocytes in vitro. In addition, Hcy-induced NADPH oxidase activity mediated the upregulation of Ref-1. Furthermore, overexpressed Ref-1 upregulated NF-kappaB and MCP-1 promoter activity, and antisense Ref-1 reduced Hcy-induced NF-kappaB DNA-binding activity and MCP-1 secretion. These data indicate that Hcy-induced ROS upregulate the expression and translocation of Ref-1 via NADPH oxidase, and then Ref-1 increases NF-kappaB activity and MCP-1 secretion in human monocytes/macrophages, which may accelerate the development of atherosclerosis.

Lipid metabolism mediated by adipocyte lipid binding protein (ALBP/aP2) gene expression in human THP-1 macrophages.

The critical initiating event in atherogenesis involves the invasion of monocytes through the endothelial wall of arteries, and their transformation from macrophages into foam cells. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) treatment, and can then be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). We previously reported that adipocyte lipid binding protein (ALBP/aP2) is a gene that is highly up-regulated in foam cells in response to oxLDL. Here, we showed that overexpression of the ALBP gene using a lentiviral construct in macrophage foam cells enhanced the accumulations of cholesterol and triglyceride, probably due to an increased expression of the scavenger receptor type AI (SR-AI), which plays an important role in cell lipid metabolism. Moreover, we determined that the expression of acyl-coenzyme A: cholesterol-acyltransferase 1 (ACAT1) gene was up-regulated by the overexpression of ALBP gene, and on the other hand, the ATP-binding cassette A1 (ABCA1) gene and hormone sensitive lipase (HSL) gene, which mediate separately cholesterol efflux and cholesterol ester hydrolysis in the macrophage cells, were down-regulated by the overexpression of ALBP gene in these cells. Finally, our data indicated that oxLDL regulates expression of ALBP related to two peroxisome proliferator-responsive elements (PPREs) which are located in ALBP promoter region. These results have determined that ALBP gene expression accelerates cholesterol and triglyceride accumulation in macrophage foam cells and affects some key gene expression for lipid metabolism, suggesting some pivotal roles of ALBP in lipid metabolism for macrophage foam cell formation.

Acyl-coenzymeA (CoA):cholesterol acyltransferase inhibition in rat and human aortic smooth muscle cells is nontoxic and retards foam cell formation.

OBJECTIVE: Studies in vitro and in vivo of macrophage foam cells have shown evidence of cytotoxicity after acyl-CoA:cholesterol acyltransferase (ACAT) inhibition. Foam cells of smooth muscle origin are also found in human and animal atherosclerotic lesions. METHODS AND RESULTS: To study whether cytotoxicity from ACAT inhibition is independent of cell type, we first established a protocol to conveniently induce aortic smooth muscle foam cell formation using cholesterol-cyclodextrin complexes (CCC). Rat aortic smooth muscle cells (ASMCs) treated for 48 hours with CCC (20 microg/mL) became foam cells by morphological (oil-red-O staining) and biochemical (approximately 1200% and approximately 180% increase in cellular esterified and free cholesterol, respectively) criteria. ACAT activity increased 500% (P<0.01 versus untreated). Similar results were obtained in human ASMC, but ACAT activity increased to an even greater extent (3200%; P<0.01 versus untreated). Western blots indicated that CCC treatment increased human (to 380+/-20% of untreated, P<0.001), but not rat, ACAT protein expression. ACAT inhibition by Fujirebio compound F1394 suppressed CCC-induced foam cell formation in rat and human ASMC, but, notably, did not induce significant cytotoxicity. CONCLUSIONS: ASMC might be more resistant to FC-induced adverse effects than are macrophages.

Transcriptional Profiling of Foam Cells Reveals Induction of Guanylate-Binding Proteins Following Western Diet Acceleration of Atherosclerosis in the Absence of Global Changes in Inflammation.

BACKGROUND: Foam cells are central to two major pathogenic processes in atherogenesis: cholesterol buildup in arteries and inflammation. The main underlying cause of cholesterol deposition in arteries is hypercholesterolemia. This study aimed to assess, in vivo, whether elevated plasma cholesterol also alters the inflammatory balance of foam cells. METHODS AND RESULTS: Apolipoprotein E-deficient mice were fed regular mouse chow through the study or were switched to a Western-type diet (WD) 2 or 14 weeks before death. Consecutive sections of the aortic sinus were used for lesion quantification or to isolate RNA from foam cells by laser-capture microdissection (LCM) for microarray and quantitative polymerase chain reaction analyses. WD feeding for 2 or 14 weeks significantly increased plasma cholesterol, but the size of atherosclerotic lesions increased only in the 14-week WD group. Expression of more genes was affected in foam cells of mice under prolonged hypercholesterolemia than in mice fed WD for 2 weeks. However, most transcripts coding for inflammatory mediators remained unchanged in both WD groups. Among the main players in inflammatory or immune responses, chemokine (C-X-C motif) ligand 13 was induced in foam cells of mice under WD for 2 weeks. The interferon-inducible GTPases, guanylate-binding proteins (GBP)3 and GBP6, were induced in the 14-week WD group, and other GBP family members were moderately increased. CONCLUSIONS: Our results indicate that acceleration of atherosclerosis by hypercholesterolemia is not linked to global changes in the inflammatory balance of foam cells. However, induction of GBPs uncovers a novel family of immune modulators with a potential role in atherogenesis.

Foam cell formation inhibits growth of Chlamydia pneumoniae but does not attenuate Chlamydia pneumoniae-induced secretion of proinflammatory cytokines.

BACKGROUND: It has not yet been determined whether lipid-loaded macrophages (foam cells), a major cellular component of atherosclerotic lesions, have the capacity to support growth of Chlamydia pneumoniae and be activated to secrete proinflammatory cytokines in response to C pneumoniae infection. METHODS AND RESULTS: Lipid loading of RAW 264.7 cells and mouse peritoneal macrophages with either oxidized or acetylated LDL significantly inhibits the growth of C pneumoniae. Modified forms of LDL are not directly toxic to C pneumoniae and do not inhibit either the initial binding or internalization of C pneumoniae by macrophages. Lipid loading does not reduce infection of macrophages with Chlamydia trachomatis. Treatment of lipid-loaded macrophages with live, heat-killed, or UV-inactivated C pneumoniae stimulates secretion of cytokines. C pneumoniae also induces expression of the mRNA for tumor necrosis factor-alpha in foam cells despite inhibition of nuclear factor-kappaB binding to DNA by prior treatment with oxidized LDL. CONCLUSIONS: Foam cell formation is not conducive to growth of C pneumoniae but does not inhibit the C pneumoniae-induced secretion of proinflammatory cytokines.