C-reactive protein levels are associated with the progression of atherosclerotic lesions in rabbits

Elevated plasma levels of C-reactive protein (CRP) are associated with increased risk of cardiovascular disease. CRP immunoreactive protein is also detected in the lesions of atherosclerosis. However, it is not known whether the CRP contents of atherosclerotic lesions are associated with the initiation and progression of atherosclerosis. To examine this hypothesis, we investigated different types of atherosclerotic lesions of rabbits fed with a cholesterol-rich diet for 6, 12, 16, and 28 weeks and examined their relationship with CRP. We measured the aortic atherosclerotic area, macrophages, and smooth muscle cells along with CRP contents in the lesions. Atherosclerotic lesions of aortas began to form at 6 weeks and were characterized by accumulation of macrophages in the intima, and lesions became more fibrotic in the advanced stage. Both plasma CRP levels and the lesional CRP contents were associated with the lesion size. Our results suggest that plasma CRP, as well as lesional CRP, associated with the formation and progression of atherosclerotic lesions.     

Arterial fatty acid-binding protein activity associated with dietarily-induced and spontaneously occurring atherosclerosis in the rabbit (Oryctolagus cuniculus)

1. Adult WHHL rabbits, or New Zealand rabbits fed either a stock chow diet or a high cholesterol diet were evaluated to assess the relationship between the development of aortic atherosclerosis and arterial FABP activity. 2. Aortic FABP activity was significantly (P less than 0.05) lower in atherosclerotic New Zealand aortas (0.039 +/- 0.008 nmol palmitoyl CoA bound/mg soluble prot) which had developed macroscopic lesions on 80% of the aortic surface as compared to lesion-free New Zealand aortas (0.053 +/- 0.002 nmol palmitoyl CoA bound/mg soluble prot). 3. In spontaneously hyperlipidemic rabbit (WHHL) aortas, FABP activity (0.023 +/- 0.004 nmol palmitoyl CoA bound/mg soluble prot) was significantly lower (P less than 0.05) than in either the normal or atherosclerotic New Zealand aortas. 4. To our knowledge, this study is the first to report a change in arterial FABP with the atherogenic process.     

Early Atherosclerotic Plaques in the Aorta Following Cytomegalovirus Infection of Mice

We show here that BALB/c mice inoculated with murine cytomegalovirus (MCMV) express viral antigens in the endothelial and smooth muscle cells of the aortic wall, and that accumulation of inflammatory cells in the aortic lumen, similar to that seen in early atherosclerotic lesions in humans, colocalizes with the site of virus antigen expression. Immunosuppression of the mice at the time of virus infection increased the expression of viral antigens and the size of early atherosclerotic lesions in the intima. The percentage of the low-density lipoprotein cholesterol (LDL-C), the major lipid contributor to atherosclerotic plaques, was significantly increased in the serum of MCMV-infected mice, whether or not the mice were fed a high cholesterol diet. Human cytomegalovirus (HCMV) significantly increased the esterified cholesterol component of the total cholesterol in a human arterial smooth muscle cell line infected in vitro with HCMV. These results suggest that CMV infection is involved in two of the major mechanisms that lead to development of atherosclerosis, i.e., immune injury and high LDL-C.     

Novel role of ADAMTS-5 protein in proteoglycan turnover and lipoprotein retention in atherosclerosis

Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE(-/-)) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE(441)) and aggrecan ((374)ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE(441) versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.     

Dendritic Cells and Regulatory T Cells in Atherosclerosis

Although macrophages and other immune system cells, especially T cells, have been shown to play disease-promoting roles in atherosclerosis, less is known about the role of antigen presenting cells. Functional, immune stimulating dendritic cells (DCs) have recently been detected in aortic intima, the site of origin of atherosclerosis. We had compared DCs with macrophages in mice with experimental atherosclerosis, to clearly define cell types by developmental and functional criteria. This review summarizes recent advances in studies of DCs in humans and in mouse models of atherosclerosis, as well as providing a simple strategy to measure regulatory T (Treg) cells in the mouse aorta.     

Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice

Several clinical and angiographic intervention trials have shown that fibrate treatment leads to a reduction of the coronary events associated to atherosclerosis. Fibrates are ligands for peroxisome proliferator-activated receptor alpha (PPARalpha) that modulate risk factors related to atherosclerosis by acting at both systemic and vascular levels. Here, we investigated the effect of treatment with the PPARalpha agonist fenofibrate (FF) on the development of atherosclerotic lesions in apolipoprotein (apo) E-deficient mice and human apoA-I transgenic apoE-deficient (hapoA-I Tg x apoE-deficient) mice fed a Western diet. In apoE-deficient mice, plasma lipid levels were increased by FF treatment with no alteration in the cholesterol distribution profile. FF treatment did not reduce atherosclerotic lesion surface area in the aortic sinus of 5-month-old apoE-deficient mice. By contrast, FF treatment decreased total cholesterol and esterified cholesterol contents in descending aortas of these mice, an effect that was more pronounced in older mice exhibiting more advanced lesions. Furthermore, FF treatment reduced MCP-1 mRNA levels in the descending aortas of apoE-deficient mice, whereas ABCA-1 expression levels were maintained despite a significant reduction of aortic cholesterol content. In apoE-deficient mice expressing a human apoA-I transgene, FF increased human apoA-I plasma and hepatic mRNA levels without affecting plasma lipid levels. This increase in human apoA-I expression was accompanied by a significant reduction in the lesion surface area in the aortic sinus. These data indicate that the PPARalpha agonist fenofibrate reduces atherosclerosis in these animal models of atherosclerosis.     

Role of dietary lipids in arteriosclerosis in experimental animals

Effects of dietary fats include the development of arteriosclerosis in humans and experimental animals, in addition to hypercholesterolemia. None of the preceding studies explicitly compared the effects of individual fatty acids. To address these issues, we chose exogenously hypercholesterolemic (ExHC) rats and apolipoprotein (apo) E deficient mice as a model for atherosclerosis and assessed the individual role of fatty acids in animals' susceptibility to atherosclerosis. The rats fed on the diet containing DHA or EPA, compared with those fed on the safflower oil (SO) diet, lowered serum cholesterol concentration, prevented platelet aggregation and slowed thickening in the ascending aorta. Apo E deficient mice developed hypercholesterolemia and severe lesion area in aortic root and arch, to a similar extent when they received DHA or SO. These results suggest a direct action of polyunsaturated fatty acids in the arterial wall, in addition to their effects on hypocholesterolemic and haemodynamic action.     

Alpha-lipoic acid attenuates atherosclerotic lesions and inhibits proliferation of vascular smooth muscle cells through targeting of the Ras/MEK/ERK signaling pathway

An infectious burden has been suggested to be associated with atherosclerosis in humans, based on the shared and underlying inflammatory responses during infection and atherosclerosis. However, the efficacy of anti-atherogenic drugs is yet to be tested against atherosclerosis in a scenario involving an infectious burden. We have examined alpha-lipoic acid (ALA) for anti-atherogenic effects in a hypercholesterolemic diet-induced atherosclerotic mouse model with inflammatory stimulation. C57BL/6 mice were fed with a hypercholesterolemic diet for 12 weeks to induce atherosclerosis. Lipopolysaccharide was intraperitoneally injected for the 1st week of study to simulate underlying infectious burden during development of atherosclerosis. ALA treatment alleviated atherosclerotic pathologies and reduced serum cholesterol and inflammatory cytokines. Consistently, atherosclerotic markers were improved by ALA treatment. In addition, ALA attenuated the proliferation and migration of vascular smooth muscle cells upon platelet-derived growth factor stimulation through the targeting of the Ras-MEK1/2-ERK1/2 pathway. This study demonstrates the efficacy of ALA on atherosclerosis with immunological complication, by showing that ALA modulates multiple pathogenic aspects of atherosclerosis induced by a hypercholesterolemic diet with inflammatory stimulation consisting of hypercholesterolemia, inflammation and VSMC activation.     

Macrophage-derived MMP-9 enhances the progression of atherosclerotic lesions and vascular calcification in transgenic rabbits

Matrix metalloproteinase-9 (MMP-9), or gelatinase B, has been hypothesized to be involved in the progression of atherosclerosis. In the arterial wall, accumulated macrophages secrete considerable amounts of MMP-9 but its pathophysiological functions in atherosclerosis have not been fully elucidated. To examine the hypothesis that macrophage-derived MMP-9 may affect atherosclerosis, we created MMP-9 transgenic (Tg) rabbits to overexpress the rabbit MMP-9 gene under the control of the scavenger receptor A enhancer/promoter and examined their susceptibility to cholesterol diet-induced atherosclerosis. Tg rabbits along with non-Tg rabbits were fed a cholesterol diet for 16 and 28 weeks, and their aortic and coronary atherosclerosis was compared. Gross aortic lesion areas were significantly increased in female Tg rabbits at 28 weeks; however, pathological examination revealed that all the lesions of Tg rabbits fed a cholesterol diet for either 16 or 28 weeks were characterized by increased monocyte/macrophage accumulation and prominent lipid core formation compared with those of non-Tg rabbits. Macrophages isolated from Tg rabbits exhibited higher infiltrative activity towards a chemoattractant, MCP-1 in vitro and augmented capability of hydrolysing extracellular matrix in granulomatous tissue. Surprisingly, the lesions of Tg rabbits showed more advanced lesions with remarkable calcification in both aortas and coronary arteries. In conclusion, macrophage-derived MMP-9 facilitates the infiltration of monocyte/macrophages into the lesions thereby enhancing the progression of atherosclerosis. Increased accumulation of lesional macrophages may promote vascular calcification.     

Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages

Foam cells formed from receptor-mediated uptake of lipoprotein cholesterol by macrophages in the arterial intima are critical in the initiation, progression, and stability of atherosclerotic lesions. Macrophages accumulate cholesterol when conditions favor esterification by acyl-CoA:cholesterol acyltransferase (ACAT) over cholesteryl-ester hydrolysis by a neutral cholesteryl-ester hydrolase, such as hormone-sensitive lipase (HSL), and subsequent cholesterol efflux mediated by extracellular acceptors. We recently made stable transfectants of a murine macrophage cell line, RAW 264.7, that overexpressed a rat HSL cDNA and had a 5-fold higher rate of cholesteryl-ester hydrolysis than control cells. The current study examined the effect of macrophage-specific HSL overexpression on susceptibility to diet-induced atherosclerosis in mice. A transgenic line overexpressing the rat HSL cDNA regulated with a macrophage-specific scavenger receptor promoter-enhancer was established by breeding with C57BL/6J mice. Transgenic peritoneal macrophages exhibited macrophage-specific 7-fold overexpression of HSL cholesterol esterase activity. Total plasma cholesterol levels in transgenic mice fed a chow diet were modestly elevated 16% compared to control littermates. After 14 weeks on a high-fat, high-cholesterol diet, total cholesterol increased 3-fold, with no difference between transgenics and controls. However, HSL overexpression resulted in thicker aortic fatty lesions that were 2.5-times larger in transgenic mice. HSL expression in the aortic lesions was shown by immunocytochemistry. Atherosclerosis was more advanced in transgenic mice exhibiting raised lesions involving the aortic wall, along with lipid accumulation in coronary arteries occurring only in transgenics. Thus, increasing cholesteryl-ester hydrolysis, without concomitantly decreasing ACAT activity or increasing cholesterol efflux, is not sufficient to protect against atherosclerosis. hormone-sensitive lipase overexpression in macrophages.     

Immunohistochemical study of the phosphorylated and activated form of c-Jun NH2-terminal kinase in human aorta

c-Jun NH₂-terminal kinase is a key enzyme mediating the cellular response to a variety of extracellular stimuli. In the present study, we performed immunohistochemical studies of the expression of the phosphorylated form of the kinase in 51 human aortas of various ages. The phosphorylated kinase immunoreactivity was strongly detected in vascular smooth muscle cells of the medial vessel layer of atherosclerotic lesions from adults. Immunoreactivity was also strongly detected in similar cells of the intima. On the other hand, immunoreactive phosphorylated kinase was only weakly detected in the medial vascular smooth muscle cells of non-atherosclerotic lesions from adults. We also investigated the expression of the phosphorylated kinase in infant aortas. In contrast to its weak immunoreactivity in adult non-atherosclerotic lesions, the kinase immunoreactivity was detected in high amounts in vascular smooth muscle cells of non-atherosclerotic lesions from infants. Thus, the abundant expression of the phosphorylated kinase in these cells in atherosclerotic lesions of adults and non-atherosclerotic lesions of infants suggests that the activation of c-Jun NH₂-terminal kinase may be an important element initiating the proliferation of vascular smooth muscle cells during atherogenesis and aortic development.     

Metabolism of sphingolipids by normal and atherosclerotic aorta of squirrel monkeys

We studied the synthesis and hydrolysis of sphingomyelin by homogenates of aortic intima plus inner media from normal squirrel monkeys and from monkeys with nutritionally-induced atherosclerosis (6-10 mo on a semi-purified diet containing butter and cholesterol). The concentrations of sphingomyelin in the aortas and plasmas of the atherosclerotic monkeys were higher than those for the normal monkeys. Palmitoyl-1-(14)C coenzyme A was actively utilized for the synthesis of ceramide (N-palmitoyl sphingosine). The addition of sphingosylphosphorylcholine increased the utilization of palmitoyl CoA in sphingomyelin synthesis, and the addition of psychosine (sphingosyl galactoside) increased the incorporation of palmitate into cerebrosides. Rates of sphingomyelin and ceramide synthesis were significantly higher in the atherosclerotic than in the control aortas. Hydrolysis of labeled sphingomyelin to ceramide was also increased in homogenates of the atherosclerotic aortas. Labeled sphingomyelin was taken up from plasma by everted carotid arteries, and this process was also enhanced by atherosclerosis. Increased rates of synthesis and of uptake from plasma of sphingomyelin may account for the increased concentrations of sphingomyelin in the atherosclerotic arteries, even though the ability to degrade sphingomyelin is also enhanced in the atherosclerotic aorta.     

Depletion of B2 but not B1a B cells in BAFF receptor-deficient ApoE mice attenuates atherosclerosis by potently ameliorating arterial inflammation

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE(-/-) mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE(-/-) mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R(-/-) ApoE(-/-) (BaffR.ApoE DKO) and BAFF-R(+/+)ApoE(-/-) (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE(-/-) mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.     

Endothelin-converting enzyme-1 increases in atherosclerotic mice: potential role of oxidized low density lipoproteins

The aim of our study was to analyze the relationships between atherosclerosis and endothelin-converting enzyme-1 (ECE-1). Four-week-old C57BL/6J [wild-type (WT)] and apolipoprotein E-deficient (apoE) mice were fed with a standard or Western-type fat diet for 8 wks. ApoE showed atherosclerotic lesions in the aorta, higher blood pressure and vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein content than WT. ApoE showed a significant increase in ECE-1 protein content and mRNA expression in aorta, lung, and kidney, without changes in heart. When an ECE-1 inhibitor, FR-901533, was administered to them, blood pressure decreased in apoE on fat diet versus apoE on normal diet and WT. ECE-1 and LOX-1 protein content were elevated in peripheral blood mononuclear cells (PBMC) from hypercholesterolemic patients. In order to study the mechanism involved in this ECE-1 up-regulation, bovine aortic endothelial cells (BAEC) were treated with oxidized-low density lipoproteins (oxLDL). OxLDL, but not LDL, increased ECE-1 protein content, mRNA expression and promoter activity. Our results demonstrate that ECE-1 increases in different atherosclerosis situations. Up-regulation of ECE-1 could contribute, at least partially, to the development of hypertension seen in apoE mice, because FR-901533 avoided it. Probably, atherosclerotic situations course with an increase of oxLDL, which is able to induce ECE-1 expression with the subsequent potential pathological effects.     

Diet‐induced early‐stage atherosclerosis in baboons: Lipoproteins,atherogenesis, and arterial compliance

Background

The purpose of this study was to determine whether dietary manipulation can reliably induce early‐stage atherosclerosis and clinically relevant changes in vascular function in an established, well‐characterized non‐human primate model.

Methods

We fed 112 baboons a high‐cholesterol, high‐fat challenge diet for two years. We assayed circulating biomarkers of cardiovascular disease (CVD) risk, at 0, 7, and 104 weeks into the challenge; assessed arterial compliance noninvasively at 104 weeks; and measured atherosclerotic lesions in three major arteries at necropsy.

Results

We observed evidence of atherosclerosis in all but one baboon fed the two‐year challenge diet. CVD risk biomarkers, the prevalence, size, and complexity of arterial lesions, plus consequent arterial stiffness, were increased in comparison with dietary control animals.

Conclusions

Feeding baboons a high‐cholesterol, high‐fat diet for two years reliably induces atherosclerosis, with risk factor profiles, arterial lesions, and changes in vascular function also seen in humans.     

Evidence that dendritic cells infiltrate atherosclerotic lesions in apolipoprotein E-deficient mice

Earlier we reported that atherosclerotic lesions of apoE-deficient mice contained cells which stained positively with anti-S-100 antibody and that cells exhibiting the ultrastructural features of dendritic cells were present in the aortic lesions. These observations suggested that dendritic cells might be involved in mouse atherosclerosis. By employing DEC-205 and MIDC-8 antibodies specific for dendritic cells, the present study has established that dendritic cells indeed accumulate in atherosclerotic lesions of apoE-deficient mice. Finding dendritic cells infiltrating atherosclerotic lesions in apoE-deficient mice offers the possibility of investigating the migratory routes of dendritic cells and their involvement in T-cell activation.     

Role of superoxide and angiotensin II suppression in salt-induced changes in endothelial Ca2+ signaling and NO production in rat aorta

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or changed to a high-salt (HS) diet (4% NaCl) for 3 days. Increases in intracellular Ca2+ ([Ca2+]i) in response to methacholine (10 microM) and histamine (10 microM) were significantly attenuated in aortic endothelial cells from rats fed a HS diet, whereas thapsigargin (10 microM)-induced increases in [Ca2+]i were unaffected. Methacholine-induced nitric oxide (NO) production was eliminated in endothelial cells of aortas from rats fed a HS diet. Low-dose ANG II infusion (5 ng x kg(-1) x min(-1) iv) for 3 days prevented impaired [Ca2+]i signaling response to methacholine and histamine and restored methacholine-induced NO production in aortas from rats on a HS diet. Adding Tempol (500 microM) to the tissue bath to scavenge superoxide anions increased NO release and caused N(omega)-nitro-L-arginine methyl ester-sensitive vascular relaxation in aortas from rats fed a HS diet but had no effect on methacholine-induced Ca2+ responses. Chronic treatment with Tempol (1 mM) in the drinking water restored NO release, augmented vessel relaxation, and increased methacholine-induced Ca2+ responses significantly in aortas from rats on a HS diet but not in aortas from rats on a LS diet. These findings suggest that 1) agonist-induced Ca2+ responses and NO levels are reduced in aortas of rats on a HS diet; 2) increased vascular superoxide levels contribute to NO destruction, and, eventually, to impaired Ca2+ signaling in the vascular endothelial cells; and 3) reduced circulating ANG II levels during elevated dietary salt lead to elevated superoxide levels, impaired endothelial Ca2+ signaling, and reduced NO production in the endothelium.     

Ultrastructural and immunohistochemical study of experimental atherosclerosis in Japanese quails

A marked plaque was produced at the tunica intima of the ascending aorta in all of the Japanese quails of 9 weeks old which fed on atherogenic diet containing 2% cholesterol for 8 weeks, while no structural changes of aortic wall were observed in Japanese quails which fed on normal basic food for the same period. The media of aorta in normal quails consist of smooth muscle cells, myofibroblast-like cells (MF) cells), and many successive elastic membranes. At the atherosclerotic lesion, many MF cells migrated from media into intima, and a part of smooth muscle cells were also differentiated to MF cells. Moreover, the most migrating MF cells differentiated to foam cells at the intimal thickness regions, and a few other MF cells also differentiated into endothelial cells of newly forming capillaries. By the immunohistochemical stainings, medial smooth muscle cells were negatively stained with anti-vimentin antibody, and the majority of cells in the intima (MF cells, foam cells, and endothelial cells) contained vimentin filaments. These results indicate that MF cells play a very important role in the development of atherosclerosis in Japanese quail. The morphologicals study offers some new insights into the evaluation of Japanese quails as an animal model of atherosclerosis.     

IL-17A is proatherogenic in high-fat diet-induced and Chlamydia pneumoniae infection-accelerated atherosclerosis in mice

The role of IL-17 in atherogenesis remains controversial. We previously reported that the TLR/MyD88 signaling pathway plays an important role in high-fat diet as well as Chlamydophila pneumoniae infection-mediated acceleration of atherosclerosis in apolipoprotein E-deficient mice. In this study, we investigated the role of the IL-17A in high-fat diet (HFD)- and C. pneumoniae-induced acceleration of atherosclerosis. The aortic sinus plaque and aortic lesion size and lipid composition as well as macrophage accumulation in the lesions were significantly diminished in IL-17A(-/-) mice fed an HFD compared with wild-type (WT) C57BL/6 control mice. As expected, C. pneumoniae infection led to a significant increase in size and lipid content of the atherosclerotic lesions in WT mice. However, IL-17A(-/-) mice developed significantly less acceleration of lesion size following C. pneumoniae infection compared with WT control despite similar levels of blood cholesterol levels. Furthermore, C. pneumoniae infection in WT but not in IL-17A(-/-) mice was associated with significant increases in serum concentrations of IL-12p40, CCL2, IFN-γ, and numbers of macrophages in their plaques. Additionally, in vitro studies suggest that IL-17A activates vascular endothelial cells, which secrete cytokines that in turn enhance foam cell formation in macrophages. Taken together, our data suggest that IL-17A is proatherogenic and that it plays an important role in both diet-induced atherosclerotic lesion development, and C. pneumoniae infection-mediated acceleration of atherosclerotic lesions in the presence of HFD.     

Method for detection and isolation of cholesteryl ester-containing "foam" cells using flow cytometry

Accumulation of cholesteryl ester within vascular cells is a defining characteristic of atherosclerotic lesions. Therefore, it is of interest to be able to monitor this critical event in the development of atherosclerosis. With this objective in mind, we have developed a method for the detection of cholesteryl ester-containing cells (i.e., foam cells) in cell suspensions prepared from enzymatically dissociated aortas. Cholesteryl ester in aortic cells was selectively stained with the fluorescent dye filipin. Because filipin binds to unesterified cholesterol but not to esterified cholesterol, it was necessary first to remove unesterified cholesterol from cells by ethanol extraction so that its presence would not interfere with the specific detection of cholesteryl ester. Then unesterified cholesterol made available by enzymatic hydrolysis of cellular cholesteryl ester could be specifically stained with filipin. The filipin-stained cell suspensions were analyzed using flow cytometry. With a flow cytometer it was possible to detect and sort cholesteryl ester-containing cells onto glass slides for microscopic analysis. Cell suspensions prepared from either grossly normal or atherosclerotic swine aortas contained cells with cholesteryl ester inclusions. As expected, these cells were more numerous in the atherosclerotic aortas. Cells with higher levels of fluorescence contained more numerous cholesteryl ester inclusions. Flow cytometric detection of cholesteryl ester-containing cells should be generally useful in studies of cellular cholesterol metabolism as well as in specific studies of cellular cholesterol accumulation in atherosclerotic vessels.