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Ambient air pollution exposure leads to increased cardiovascular morbidity and mortality. Previous studies support a causal link between particulate matter (PM) exposure and cardiometabolic effects including atherosclerosis. We have previously reported that whole diesel exhaust (DE) exposure for 16 weeks led to increased hepatic triglycerides (TG), 9- and 13-hydroxyoctadecadienoic acid (HODEs), plasma cholesterol and TG in apolipoprotein E -/- (ApoE) mice. Furthermore, gut microbiota composition was altered in mice exposed to ambient ultrafine particles by oral gavage for 10-weeks, that significantly associated with atherogenic lipid metabolites. Thus, we hypothesized that chronic exposure to DE leads to dysbiosis that could likely mediate oxidative and metabolic effects in the liver. Male ApoE -/- mice at 31 weeks of age were randomly assigned to two groups (n=10/group) exposed to either DE or filtered air (FA) for 16 weeks (6 hours/day, 5 days/week). 16S sequencing of the cecum exhibited significant differences in the abundance of bacterial taxa, as well as alpha and beta diversity indices of the DE-exposed gut microbiome vs. FA (p

Uterine stress is associated with an increased risk of later life metabolic diseases. In this study, we investigated the effect of diesel exhaust (DE) exposure in utero on adult susceptibility to atherosclerosis in genetically hyperlipidemic mice. Pregnant apolipoprotein E-deficient mice received either DE exposure (~250–300 μg/m3 PM2.5 for 6 h/day, 5 days/week) or filtered air (FA) throughout gestation. Treatment effects on litter size and gender distribution were recorded. Plasma cholesterol and triglycerides were measured at 8, 12 and 16 weeks of age. Urinary 8-isoprostane and liver 8-hydroxy-deoxyguanosine levels were measured at killing at 16 weeks of age. Expression of the antioxidant genes heme oxygenase-1 and the glutamate-cysteine ligase modifier and catalytic subunits were measured in the lung, liver and aorta. The average area and frequency of atherosclerotic lesions were measured in the aortic sinus and innominate arteries. There were significantly smaller litters and higher postnatal mortality in the DE-exposed mice. There were no significant differences in plasma lipids or lipoprotein profiles, expression of antioxidant genes or markers of oxidative stress between treatment groups. There were also no significant differences in average atherosclerotic lesion area in the aortic sinus or innominate arteries of the DE and FA groups although there was a higher frequency of lesions in the DE-exposed group. Our study indicates that in utero DE exposure does not influence later life lipoprotein metabolism, redox homeostasis or the risk of developing larger atherosclerotic lesions.

Introduction: Air pollution associates with increased cardiovascular morbidity and mortality, partly due to induction of dyslipidemia and metabolic syndrome. Our goal was to dissect mechanisms involved. Hypothesis: Diesel exhaust exposure induces hyperlipidemia and liver steatosis by dysregulating lipid metabolism, and altering gut microbiota composition. Methods: We assessed the effects of exposure to air pollution on lipid metabolism in mice through assessment of plasma lipids and lipoproteins, oxidized fatty acids 9-HODE and 13-HODE, lipid and carbohydrate metabolism, and gut microbiota composition. Findings were corroborated and mechanisms further assessed in culture of HepG2 hepatocytes. Results: ApoE KO mice exposed to inhaled diesel exhaust (DE, 6 hours/day, 5 days/week for 16 weeks) exhibited significantly (p< 0.05) elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and increased hepatic levels of 9-HODE and 13-HODE (oxidative products of linoleic acid) indicative of increased oxidative stress, as compared with control mice treated with filtered air (FA). DE also led to downregulation of Acad9 , which suggested decreased β-oxidation of fatty acids and decreased lipid catabolism. A direct effect of DEP exposure on hepatocytes was demonstrated by treatment of HepG2 cells with a methanol extract of DE particles followed by loading with oleic acid. As observed in vivo , this led to decreased ACAD9 expression, increased triglyceride content, and altered total, mitochondrial and ATP-linked respiration assessed by a Seahorse metabolic flux analyzer, indicative of mitochondrial dysfunction. Direct treatment of mitochondrias with DE particles affected mitochondrial complexes I and IV. Conclusions: Diesel exhaust exposure leads to dyslipidemia and liver steatosis in ApoE KO mice, likely due to mitochondrial dysfunction and decreased lipid catabolism.

Introduction: Previous animal studies have shown that long exposures to particulate matter (PM) and diesel exhaust particles (DEP) promote atherosclerosis. Air pollutants are also reported to cause nonalcoholic steatohepatitis (NASH), liver fibrosis and insulin resistance in animals exposed to PM Methods: ApoE null mice were briefly exposed to diesel exhaust (DE) or filtered air (FA) to assess liver content of lipids, lipid and carbohydrate metabolism. Illumina microarrays and metabolomic analysis were performed in liver tissue. Transcriptomic data was analyzed using the Illumina Beadstudio software. Gluconeogenesis and glycogenolysis were evaluated in HepG2 cells after treatment with diesel exhaust particles (DEP). Gene expression was determined by qPCR. Results: DE exposures led to a significant increase in both liver triglyceride (Mean ± SEM, FA: 0.16 ± 0.01 vs DE: 0.22 ± 0.02 mg/mg protein, n=7, p=0.02) and cholesterol (FA: 15.53 ± 1.53 vs DE: 23.26 ± 2.16 μg/mg protein, n=5, p=0.02) content in ApoE null mice. Microarray analysis depicted a dysregulation of 477 genes, and metabolomic analysis identified 70 metabolites significantly upregulated in the DE group while 48 metabolites were downregulated. Interestingly, metabolites in the citric acid cycle demonstrated significant changes after DE exposure likely due to mitochondrial dysfunction. Key driver analysis revealed Pck1 as a key driver gene. In-vitro experiments in HepG2 cells treated with DEP exhibited a significant upregulation in PCK1 mRNA expression (DEP: 7.31 ± 0.2 vs control: 1.07 ± 0.07, n=3, p Conclusion: Exposure to diesel exhaust leads to multiple alterations in carbohydrate and lipid metabolism including increased glycogenolysis, gluconeogenesis and PCK1 upregulation.

Macrophages are prominent cells in acute and chronic inflammatory diseases. Recent studies highlight a role for macrophage proliferation post-monocyte recruitment under inflammatory conditions. Using an acute peritonitis model, we identify a significant defect in macrophage proliferation in mice lacking the leukocyte transmembrane protease ADAM17. The defect is associated with decreased levels of macrophage colony-stimulating factor 1 (CSF-1) in the peritoneum and is rescued by intraperitoneal injection of CSF-1. Cell surface CSF-1 (csCSF-1) is one of the substrates of ADAM17. We demonstrate that both infiltrated neutrophils and macrophages are major sources of csCSF-1. Furthermore, acute shedding of csCSF-1 following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the neutrophil surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages and to prevent macrophage proliferation. In acute inflammation, csCSF-1 release and macrophage proliferation are self-limiting due to transient leukocyte recruitment and temporally restricted csCSF-1 expression. In chronic inflammation, such as atherosclerosis, the ADAM17-mediated lesional macrophage proliferative response is prolonged. Our results demonstrate a novel mechanism whereby ADAM17 promotes macrophage proliferation in states of acute and chronic inflammation.

Recent studies highlight a role for macrophage proliferation post monocyte recruitment in inflammatory conditions, such as atherosclerosis. However, the mechanisms regulating macrophage proliferation are not well understood. Using an acute peritonitis model, we identified a 40 ± 8% reduction (mean ± SEM; n=5; p

BACKGROUND AND AIMS: Recent in vitro studies have showed that in macrophages deletion of the non-selective Ca(2+)-permeable channel TRPC3 impairs expression of the osteogenic protein BMP-2. The pathophysiological relevance of this effect in atherosclerotic plaque calcification remains to be determined. METHODS: We used Ldlr(−/−) mice with macrophage-specific loss of TRPC3 (MacTrpc3(−/−)/Ldlr(−/−)) to examine the effect of macrophage Trpc3 on plaque calcification and osteogenic features in advanced atherosclerosis. RESULTS: After 25 weeks on high fat diet, aortic root plaques in MacTrpc3(−/−)/Ldlr(−/−) mice showed reduced size, lipid and macrophage content compared to controls. Plaque calcification was decreased in MacTrpc3(−/−)/Ldlr(−/−) mice, and this was accompanied by marked reduction in BMP-2, Runx-2 and phospho-SMAD1/5 contents within macrophage-rich areas. Expression of Bmp-2 and Runx-2 was also reduced in bone marrow-derived macrophages from MacTrpc3(−/−)/Ldlr(−/−) mice. CONCLUSIONS: These findings show that, in advanced atherosclerosis, selective deletion of TRPC3 in macrophages favors plaque regression and impairs the activity of a novel macrophage-associated, BMP-2-dependent mechanism of calcification.

Objective: Chronic kidney disease (CKD) greatly increases the risk of cardiovascular disease morbidity and mortality. Mounting data has identified the receptor activator of NF-κB (RANK) pathway as an important mediator of the accelerated atherosclerosis and vascular calcification observed in CKD. Here we investigated the role of myeloid RANK in a model of CKD accelerated atherosclerosis and vascular calcification in apolipoprotein E-deficient mice (apoE -/- ). Methods and Results: We used the Lys M promoter to conditionally delete granulocyte and monocyte RANK in atherogenic apoE -/- mice. To induce CKD RANK (flox/flox) LysM (cre/+) Apoe (-/-) (apoE ΔRANK ) and LysM (cre/+) Apoe (-/-) (apoE CTL ) underwent a two-step surgical procedure to achieve 5/6 th nephrectomy. Additional control animals underwent sham surgeries. Blood urea nitrogen (BUN) levels were elevated approximately two-fold in the CKD mice relative to sham-operated mice regardless of genotype. CKD was accompanied by a trend toward lower body weight, as well as slightly elevated circulating phosphate levels at the time of sacrifice. CKD induced a 47% elevation of plasma cholesterol levels in the apoE ΔRANK and apoE CTL mice, with no significant difference in cholesterol levels between the apoE ΔRANK and apoE CTL CKD mice. Interestingly, CKD resulted in elevated triglycerides in apoE ΔRANK mice, but not in apoE CTL mice. CKD mice age 22 and 34 weeks had larger average lesional cross-section areas in the root of the aorta as compared to sham operated mice (pΔRANK mice were significantly larger than those from apoE CTL CKD mice. In all genotypes and disease conditions we observed very little vascular calcification. Conclusions: These results suggest an atheroprotective role for myeloid RANK signaling in CDK-dependent lesion acceleration.

Aims The goal of this study was to determine whether the A1 adenosine receptor (AR) plays a role in atherosclerosis development and to explore its potential mechanisms. Methods and results Double knockout (DKO) mice, deficient in the genes encoding A1 AR and apolipoprotein E (apoE), demonstrated reduced atherosclerotic lesions in aortic arch (en face), aortic root, and innominate arteries when compared with apoE-deficient mice (APOE-KO) of the same age. Treating APOE-KO with an A1 AR antagonist (DPCPX) also led to a concentration-dependent reduction in lesions. The total plasma cholesterol and triglyceride levels were not different between DKO and APOE-KO; however, higher triglyceride was observed in DKO fed a high-fat diet. DKO also had higher body weights than APOE-KO. Plasma cytokine concentrations (IL-5, IL-6, and IL-13) were significantly lower in DKO. Proliferating cell nuclear antigen expression was also significantly reduced in the aorta from DKO. Despite smaller lesions in DKO, the composition of the innominate artery lesion and cholesterol loading and efflux from bone marrow-derived macrophages of DKO were not different from APOE-KO. Conclusion The A1 AR may play a role in the development of atherosclerosis, possibly due to its pro-inflammatory and mitogenic properties.

Objective: Vascular calcification is highly correlated with cardiovascular disease morbidity and mortality. Osteoprotegerin (OPG) is a secreted decoy receptor for receptor activator of NF-κB ligand (RANKL). Inactivation of OPG in apolipoprotein E-deficient (ApoE-/-) mice increases lesion size and calcification. The mechanism(s) by which OPG is atheroprotective and anticalcific have not been entirely determined. We investigated whether OPG-deficient vascular smooth muscle cells (VSMCs) are more susceptible to mineralization and whether RANKL mediates this process. Results: Lesion-free aortas from 12-week-old ApoE-/-OPG-/- mice had spotty calcification, an appearance of osteochondrogenic factors and a decrease of smooth muscle markers when compared to ApoE-/-OPG+/+ aortas. In osteogenic conditions, VSMCs isolated from ApoE-/-OPG-/- (KO-VSMC) mice deposited more calcium than VSMCs isolated from ApoE-/-OPG+/+ (WT-VSMC) mice. Gene expression and biochemical analysis indicated accelerated osteochondrogenic differentiation. Ablation of RANKL signaling in KO-VSMCs rescued the accelerated calcification. While WT-VSMCs did not respond to RANKL treatment, KO-VSMCs responded with enhanced calcification and the upregulation of osteochondrogenic genes. RANKL strongly induced interleukin 6 (IL-6), which partially mediated RANKL-dependent calcification and gene expression in KO-VSMCs. Conclusions: OPG inhibits vascular calcification by regulating the procalcific effects of RANKL on VSMCs and is thus a possible target for therapeutic intervention.

Atherosclerosis is the primary cause of heart attacks and strokes and is caused by a chronic inflammatory response in the coronary and carotid arteries. A new study shows that smooth muscle cells within atherosclerotic plaques can change phenotypes to become macrophage-like cells, thereby revealing the remarkable plasticity of these cells.

Objective— Osteoprotegerin (OPG) is a decoy receptor for the osteoclast differentiation factor receptor activator of NF-κB ligand. OPG regulates bone homeostasis, and its inactivation in mice results in severe osteoporosis. OPG deficiency in apolipoprotein E (ApoE) −/− mice results in increased atherosclerotic lesion size and calcification. Furthermore, receptor activator of NF-κB ligand enhances macrophage-dependent smooth muscle cell calcification in vitro. Here, we hypothesized that reconstitution of ApoE −/− OPG −/− mice with ApoE −/− OPG +/+ bone marrow (BM) would be sufficient to rescue lesion progression and vascular calcification. Conversely, reconstitution of ApoE −/− OPG +/+ mice with ApoE −/− OPG −/− BM may accelerate lesion progression and vascular calcification. Approach and Results— ApoE −/− OPG −/− mice transplanted with ApoE −/− OPG +/+ BM developed smaller atherosclerotic lesions and deposited less calcium in the innominate artery than that of ApoE −/− OPG −/− mice transplanted with ApoE −/− OPG −/− BM. There were no differences in lesion size and calcification in ApoE −/− OPG +/+ mice transplanted with BM from ApoE −/− OPG −/− or ApoE −/− OPG +/+ mice. The large lesions observed in the ApoE −/− OPG −/− mice transplanted with OPG −/− BM were rich in chondrocyte-like cells, collagen, and proteoglycans. Importantly, the ApoE −/− OPG −/− mice transplanted with OPG +/+ BM remained osteoporotic, and the ApoE −/− OPG +/+ mice did not show signs of bone loss regardless of the type of BM received. In coculture experiments, macrophages and mesenchymal stem cells derived from ApoE −/− OPG −/− BM induced more vascular smooth muscle cell calcification than cells derived from ApoE −/− OPG +/+ mice. Conclusions— These results indicate that OPG derived either from the BM or from the vessel wall is sufficient to slow down lesion progression and vascular calcification independent of bone turnover.

Glutathione (GSH) is depleted early in the course of Parkinson’s disease (PD), and deficiency has been shown to perpetuate oxidative stress, mitochondrial dysfunction, impaired autophagy, and cell death. GSH repletion has been proposed as a therapeutic intervention. The objective of this study was to evaluate whether intranasally administered reduced GSH, (in)GSH, is capable of augmenting central nervous system GSH concentrations, as determined by magnetic resonance spectroscopy in 15 participants with mid-stage PD. After baseline GSH measurement, 200 mg (in)GSH was self-administered inside the scanner without repositioning, then serial GSH levels were obtained over ~1 h. Statistical significance was determined by one-way repeated measures analysis of variance. Overall, (in)GSH increased brain GSH relative to baseline (P

Objectives. Oxidative stress contributes to Parkinson’s disease (PD) pathophysiology and progression. The objective was to describe central and peripheral metabolites of redox metabolism and to describe correlations between glutathione (Glu) status, age, and disease severity.Methods. 58 otherwise healthy individuals with PD were examined during a single study visit. Descriptive statistics and scatterplots were used to evaluate normality and distribution of this cross-sectional sample. Blood tests and magnetic resonance spectroscopy (MRS) were used to collect biologic data. Spearman’s rank-order correlation coefficients were used to evaluate the strength and direction of the association. The Unified PD Rating Scale (UPDRS) and the Patient-Reported Outcomes in PD (PRO-PD) were used to rate disease severity using regression analysis.Results. Blood measures of Glu decreased with age, although there was no age-related decline in MRS Glu. The lower the blood Glu concentration, the more severe the UPDRS (P=0.02, 95% CI: −13.96, −1.14) and the PRO-PD (P=0.01, 95% CI: −0.83, −0.11) scores.Discussion. These data suggest whole blood Glu may have utility as a biomarker in PD. Future studies should evaluate whether it is a modifiable risk factor for PD progression and whether Glu fortification improves PD outcomes.

Chlamydia pneumoniae is an obligate intracellular pathogen that has been found in atherosclerotic lesions and chronic infection has been linked to an increased risk of cardiovascular disease. C. pneumoniae infects all of the cell types in normal and diseased blood vessels. In this chapter, we discuss the receptors that mediate the cellular uptake of C. pneumoniae and the unique intracellular developmental cycle of C. pneumoniae including conversion to a persistent phenotype. We further discuss the specific in vitro responses of vascular cells to C. pneumoniae infection with a focus on the role of toll-like receptors and induction of cytokine expression as well as effects on cellular metabolism and turnover. We also discuss the in vivo responses including the effects on vascular reactivity and atherosclerosis and include discussion of the results of clinical trials with antibiotics for reducing cardiovascular disease end points. We conclude the chapter by discussing how C. pneumoniae infection can have indirect systemic effects that could have an impact on the progression of vascular disease.

Pharmacokinetic modeling of dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) is used to noninvasively characterize neovasculature and inflammation in atherosclerotic vessels by estimating perfusion characteristics, such as fractional plasma volume vp and transfer constant Ktrans. DCE-MRI has potential to study the evolution of nascent lesions involving early pathological changes. However, currently used bright-blood DCE-MRI approaches are difficult to apply to small lesions because of the difficulty in separating the signal in the thin vessel wall from the adjacent lumen. By suppressing the lumen signal, black-blood DCE-MRI techniques potentially provide a better tool for early atherosclerotic lesion assessment. However, whether black-blood DCE-MRI can detect temporal changes in physiological kinetic parameters has not been investigated for atherosclerosis. This study of balloon-injured New Zealand White rabbits used a reference-region-based pharmacokinetic model of black-blood DCE-MRI to evaluate temporal changes in early experimental atherosclerotic lesions of the abdominal aorta. Six rabbits were imaged at 3 and 6 months after injury. Ktrans was found to increase from 0.10±0.03 min(-1) to 0.14±0.05 min(-1) (P=0.01). In histological analysis of all twelve rabbits, Ktrans showed a significant correlation with macrophage content (R=0.70, P=0.01). These results suggest black-blood DCE-MRI and a reference-region kinetic model could be used to study plaque development and therapeutic response in vivo.

Recent discoveries in the field of air pollution toxicology highlight the potential impact of specific sources of air pollution, especially related to roadway emissions, on acute and chronic cardiovascular disease. This review covers potential mechanisms, both in terms of biological pathways and chemical drivers, to explain these observations.Air pollution is associated with chronic progression of cardiovascular disease. Roadway exposures appear to have a strong correlation to these adverse outcomes. Controlled toxicological studies highlight potential interactions between vehicle-source emissions and adverse vascular outcomes. Mechanistically, a role for both innate and adaptive immune responses is emerging, with important recent findings demonstrating that immunomodulatory pattern-recognition receptors such as Toll-like receptor-4 and lectin-like oxidized LDL receptor-1 may play a role in communicating airway exposures to cardiovascular outcomes.An improved understanding of the sources and mechanisms underlying adverse cardiovascular health outcomes of air pollution would enhance our ability to manage vulnerable populations and establish precise, effective regulatory policies.

Objective— Glutamate-cysteine ligase (GCL) is the rate-limiting step in glutathione synthesis. The enzyme is a heterodimer composed of a catalytic subunit, GCLC, and a modifier subunit, GCLM. We generated apolipoprotein E (apoE)−/− mice deficient in GCLM ( apoE −/−/ Gclm −/−) and transgenic mice that overexpress GCLC specifically in macrophages ( apoE −/−/ Gclc-Tg ) to test the hypothesis that significantly altering the availability of glutathione has a measurable impact on both the initiation and progression of atherosclerosis. Methods and Results— Atherosclerotic plaque size and composition were measured in the innominate artery in chow-fed male and female mice at 20, 30, 40, and 50 weeks of age and in the aortic sinus at 40 and 50 weeks of age. The apoE −/−/ Gclm −/− mice more rapidly developed complex lesions, whereas the apoE −/−/ Gclc-Tg mice had reduced lesion development compared with the littermate apoE −/− control mice. Transplantation of bone marrow from the apoE −/−/ Gclm −/− and apoE −/−/ Gclc-Tg mice into apoE −/− mice with established lesions also stimulated or inhibited further lesion development at 30 weeks posttransplant. Conclusion— Gain and loss of function in the capacity to synthesize glutathione especially in macrophages has reciprocal effects on the initiation and progression of atherosclerosis at multiple sites in apoE−/− mice.

Traffic related particulate matter air pollution is a risk factor for cardiovascular events; however, the biological mechanisms are unclear. We hypothesize that diesel exhaust (DE) inhalation induces up-regulation of inducible nitric oxide synthase (iNOS), which is known to contribute to vascular dysfunction, progression of atherosclerosis and ultimately cardiovascular morbidity and mortality. Methods: ApoE knockout mice (30-week) were exposed to DE (at 200 {mu}g/m{sup 3} of particulate matter) or filtered-air (control) for 7 weeks (6 h/day, 5 days/week). iNOS expression in the blood vessels and heart was evaluated by immunohistochemistry and western blotting analysis. To examine iNOS activity, thoracic aortae were mounted in a wire myograph, and vasoconstriction stimulated by phenylephrine (PE) was measured with and without the presence of the specific inhibitor for iNOS (1400 W). NF-{kappa}B (p65) activity was examined by ELISA. The mRNA expression of iNOS and NF-{kappa}B (p65) was determined by real-time PCR. Results: DE exposure significantly enhanced iNOS expression in the thoracic aorta (4-fold) and heart (1.5 fold). DE exposure significantly attenuated PE-stimulated vasoconstriction by {approx} 20%, which was partly reversed by 1400 W. The mRNA expression of iNOS and NF-{kappa}B was significantly augmented after DE exposure. NF-{kappa}B activity was enhanced 2-fold after DE inhalation, andmore » the augmented NF-{kappa}B activity was positively correlated with iNOS expression (R{sup 2} = 0.5998). Conclusions: We show that exposure to DE increases iNOS expression and activity possibly via NF-{kappa}B-mediated pathway. We suspect that DE exposure-caused up-regulation of iNOS contributes to vascular dysfunction and atherogenesis, which could ultimately lead to urban air pollution-associated cardiovascular morbidity and mortality. - Highlights: > Exposed ApoE knockout mice (30-week) to diesel exhaust (DE) for 7 weeks. > Examine iNOS expression and activity in the blood vessels and heart. > DE exposure enhanced iNOS protein and mRNA expression in the aorta and heart. > iNOS activity was also increased after DE exposure. > This up-regulation of iNOS may contribute to vascular dysfunction and atherogenesis.« less

Exposure to urban air pollution is an independent risk factor for increased cardiovascular diseases. Heat shock protein 70 (HSP70) has been implicated in the pathogenesis of vascular dysfunction and cardiovascular diseases. This study has been designed to determine whether inhalation of urban air induces HSP70 expression in the lung and blood as well as the association of HSP70 and air pollution-induced vascular dysfunction. Apolipoprotein E (Apo-E) deficient mice were exposed to diesel exhaust (DE) either acutely (3 days, 200 or 400 µg/m(3) for 6 h/day) or chronically (7 weeks, 200 or 400 µg/m(3) for 6 h/day). HSP70 was measured in the lung using immunohistochemistry, and in the plasma by ELISA. Abdominal aorta rings were used to determine vascular functional responses. Chronic DE-exposure increased the fraction of HSP70 positive alveolar macrophages (AM) that was related to the fraction of particle-laden AM in the lung (r(2) = 0.48, p 0.01). Chronic DE-exposure increased plasma HSP70 levels and reduced blood vessel responses to phenylephrine (PE). The fraction of particle-laden HSP70 positive AM was associated with abnormal vasoconstriction responses to PE induced by DE-exposure (r(2) = 0.12, p = 0.02). Our results show that chronic inhalation of DE increases HSP70 expression in the lung and systemic circulation, and we postulate that HSP70 possibly contributes to air pollution induced vascular dysfunction and cardiovascular diseases.

Eur J Clin Invest 2011; 41 (11): 1164–1171 Abstract Background The multiligand receptor for advanced glycation end products (RAGE) of the immunoglobulin superfamily is expressed on multiple cell types implicated in the inflammatory response in atherosclerosis. We sought to determine the role of bone marrow-derived RAGE in different stages of atherosclerotic development in apolipoprotein E-deficient mice (apoE−/−). Methods Seven- and 23-week-old apoE−/− mice (n = 40) were lethally irradiated and given bone marrow from RAGE null (RAGE−/−/apoE−/−) or RAGE-bearing (RAGE+/+/apoE−/−) mice to apoE−/− mice to generate double knockout bone marrow chimera (RAGE−/−/apoE−/−bmc and RAGE+/+/apoE−/−bmc-, respectively). After 16 weeks on a standard chow diet, mice were sacrificed and atherosclerotic lesion formation was evaluated. Results Plaques in the aortic root of the young mice showed no significant difference in maximum plaque size (217 470 ± 17 480 μm2 for the RAGE−/−/apoE−/−bmc mice compared to 244 764 ± 45 840 μm2), whereas lesions in the brachiocephalic arteries of the older RAGE−/−/apoE−/−bmc mice had significantly smaller lesions (94 049 ± 13 0844 μm2 vs. 145 570 ± 11 488 μm2, P

The acute phase response to Chlamydia pneumoniae infection was analyzed over a 72 h period post-infection in C57BL/6J mice. A single intra-nasal inoculation stimulated statistically significant increases in the plasma levels of IL-2, IL-5, IL-6, IL-10, IL-12, GM-CSF, IFN-gamma, and serum amyloid A but not TNF-alpha, IL-1beta, IL-4 and serum amyloid P. There was also a decrease in the activity of the HDL protective enzyme paraoxonase as well as a reduced ability of HDL to prevent oxidation of palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine by hydroperoxyoctadecadienoic acid at 48 and 72 h post-infection. To determine whether the C. pneumoniae induced acute phase response had any effect on atherosclerotic plaque stability, we measured the frequency of intra-plaque hemorrhage as a marker of plaque disruption in the innominate arteries of apolipoprotein E deficient mice at 29-30 weeks and 1.5-2.0 years of age. There was an increased frequency of intra-plaque hemorrhage only in the older mice infected with the live organism (8/14) as compared to mice treated with killed C. pneumoniae (2/11) or sham inoculated with PBS (2/12). These results suggest that acute phase reactant proteins produced in response to pulmonary infection with C. pneumoniae may contribute to the progression and destabilization of atherosclerotic lesions.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Uptake of homocysteine induces oxidative stress in macrophages. Antioxidant response elements (AREs) are regulatory elements within promoters of genes, which protect cells against oxidative stress. The current study investigated whether homocysteine induces transcription of glutamate-cysteine ligase (Gcl), via ARE driven gene expression in mouse macrophages. Gcl is the rate-limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant. Gcl is heterodimeric and the genes encoding the subunits of Gcl contain several AREs within their 5'-promoter regions. Treatment of mouse macrophages with d-/l-homocysteine (50microM) induced depletion of intracellular glutathione and a compensatory increase in Gcl activity. Electro mobiliy shift assays demonstrated increased binding of nuclear proteins to ARE-containing oligonucleotides. Real-time RT-PCR revealed increased mRNA-expression of the catalytic subunit of Gcl (Gclc) after treatment with homocysteine, and this occurred via increased transcription as demonstrated with luciferase promoter reporter constructs for Gclc. Additional site directed mutagenesis demonstrated that ARE4 plays a direct role in mediating induction of Gclc by homocysteine. Supershift analysis and Western blotting revealed that Nrf2 signalling is critical in homocysteine-induced activation of ARE4. Inhibition of MAP kinase activity reduced binding of nuclear proteins to the AREs, nuclear expression of Nrf2 and mRNA expression of Gclc. Western blotting demonstrated phosporylation of ERK1/2 in homocysteine treated macrophages. These data suggest that ARE-driven gene expression of Gclc via a MEK/Nrf2 pathway could help to protect macrophages from oxidative stress due to hyperhomocysteinemia.

It is documented that hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis, but whether elevated plasma homocysteine contributes to the progression of atherosclerosis in aged animals with hypercholesterolemia is still unknown. HHcy was induced in apolipoprotein E (ApoE) knockout mice (male, 32 weeks old) by feeding 2% methionine/low folate (1 mg/kg) diet for 20 weeks. HHcy induced by methionine feeding significantly increased oxidative stress, as measured by thiobarbituric-reactive substances in livers (P.05) and genetic expression of Cu,Zn-superoxide dismutase, in methionine-fed animals compared with controls (P.05). Furthermore, lipoprotein profiles were changed, in that low-density lipoprotein-cholesterol was shifted to very low-density lipoprotein in the methionine-supplemented group. However, nuclear factor kappaB activity, atherosclerotic lesions, hepatic glutathione level, lipid profiles, and activities of aspartate aminotransferase and alanine aminotransferase were not significantly different. These findings suggest that HHcy induced by methionine may promote disturbances in lipid peroxidation and modify lipoprotein metabolism but not contribute to the progression of atherosclerotic lesion in aged ApoE knockout mice.

Chlamydia pneumoniae, a common respiratory pathogen, has been associated with cardiovascular disease. C. pneumoniae infection accelerates atherosclerotic lesion development in hyperlipidemic animals. Retinoic acid, an anti-oxidant, inhibits infection of endothelial cells by C. pneumoniae. The present study demonstrated that retinoic acid suppresses the acceleration of foam cell lesion development induced by C. pneumoniae in hyperlipidemic C57BL/6J mice. Retinoic acid treatment had no effect on foam cell lesion development in uninfected animals. Lung infection and duration was decreased in treated mice, suggesting one mechanism by which retinoic acid reduces C. pneumoniae accelerated foam cell lesion formation in hyperlipidemic mice.

The innominate artery is a predilection site for atherosclerotic lesion formation in hyperlipidemic mice. The lesions at this site in chow-fed apo E-/- mice progress from fatty streaks through stages that include atheroma with large necrotic areas, fibro-fatty nodules containing chondrocyte-like cells and highly calcified, acellular plaques. The advanced lesions in the innominate arteries of the apo E-/- mice exhibit a reproducible frequency of intra-plaque hemorrhage that occurs primarily as a result of fissures through lateral fatty streaks that form adjacent to or on top of the established plaques. However, this plaque disruption is not equivalent to plaque rupture in human lesions where there is rupture of well formed fibrous caps. The plaque disruption in the lesions of the chow-fed apo E-/- mice also do not lead to formation of occlusive thrombi, the predominant marker of plaque rupture in humans. Thus, although the lesions in the innominate arteries of hyperlipidemic mice progress to very advanced stages of the disease, they are not, in our opinion a model in which to study the mechanisms of plaque rupture in humans. The advanced lesions in the innominate arteries of the apo E-/- mice may however be adequate models for studying vascular fibrosis and calcification.

The majority of graduating US medical students reported inadequate nutrition training over the past decade. This trend could in part be due to the lack of valid measures to assess the relationship between adequacy of nutrition training and proficiency on nutrition topics deemed essential. The study's objective was to test the hypothesis that self-reported nutrition proficiency is positively correlated with the perceived adequacy (quality, quantity, coverage and importance) of nutrition training of University of Washington medical students.Cross-sectional e-mail survey of 1st to 4th year medical students (n = 708), including a survey prompt and three e-mail follow-up measures. To reduce and interpret the survey data, principal components analysis was employed, followed by Varimax rotation with Kaiser normalization. To assess internal consistency reliability, alpha (alpha) of nutrition proficiency items and factors was determined.A 44.5% response rate was achieved (n = 315 respondents). The 31-item questionnaire was reduced to 6 factors, explaining 60.2% of the total variance (alpha = 0.947). Self reported nutrition proficiency was positively correlated with the perceived quality, quantity and coverage of nutrition training in all 6 essential nutrition factors or topics determined after factor analysis (P0.01).Quality and coverage may be effective gauges of adequacy of nutrition training and related nutrition proficiency in medical education. Current national medical education evaluation measures focus on the quantity of nutrition instruction. The lowest reported proficiency topics; nutrition and disease management, micronutrients and complementary and alternative medicine are recommended for particular curricular emphasis.

Inflammatory mechanisms are involved in initiation and progression of atherosclerotic lesions. Previous studies demonstrated antiinflammatory and consecutive antiatherosclerotic effects of the adenosine analogue 3-Deazaadenosine (c(3) Ado) on early lesion development. The present study evaluated the effect of long-term administration of c(3) Ado in a mouse model of advanced atherosclerosis. Apolipoprotein E-deficient mice (age, 35 weeks; n = 31) with already established advanced atherosclerotic lesions were fed either a diet supplemented with c Ado or a regular chow diet for 21 weeks. Treatment resulted in a significant reduction of serum homocysteine levels. Lesion size and lesion morphology, such as frequency of intraplaque hemorrhage, size of necrotic cores, thickness of fibrous caps, and macrophage content within the plaque, were not different between the groups. Lesion calcification, expression of alpha-actin, and intercellular adhesion molecule-1, but not vascular cell adhesion molecule-1, were inhibited by treatment with c(3) Ado. We could not detect any effect on serum concentrations of interleukin-10 (IL-10) and interleukin-1beta (IL-1beta) or on soluble adhesion molecules intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Electromobility shift assays of protein extracts isolated from aortas did not demonstrate different binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) after treatment with c Ado. Long-term treatment with the adenosine analogue 3-Deazaadenosine did not show significant effects on progression and stability of advanced atherosclerotic lesions in older apolipoprotein E-deficient mice. A potential antiatherosclerotic effect of c(3)Ado (eg, mediated through inhibition of adhesion molecules) might therefore be limited to prevention of early lesion formation and does not seem to play a relevant role in modifying advanced atherosclerotic disease.

To be useful, animal models for human diseases must be well defined.1 Thus we are concerned that investigators will be mislead by the definitions and terminology used by Jackson et al1a to describe putative plaque rupture models in mice. We are especially concerned with their use of “acute plaque rupture” to describe murine lesions that do not mimic any of the key features of human plaque rupture and use of “buried fibrous caps” as questionable evidence for past ruptures. The consensus of cardiologists and pathologists is that rupture of human atherosclerotic lesions, defined as a structural defect in the fibrous cap overlying a necrotic core, is responsible for most coronary thrombi.2 This defect is associated with variable amounts of luminal thrombosis and plaque hemorrhage. Although neither thrombus nor plaque hemorrhage is required by this definition of plaque rupture,3 detection of these vital reactions is critically important to exclude possible post mortem artifacts. Confusingly, and in contrast to their original publications that emphasized luminal thrombosis,4,5 the current review by Jackson et al no longer considers thrombosis an important component of their “acute plaque rupture” model in mice. Interpretation of mouse models will be very confusing if terminology is used in an inconsistent fashion. Use of precise and transparent terms does not in any way limit the use of animal models to study specific processes. Death of smooth …

Michael R Preusch,1,2 Nicholas Ieronimakis,1 Errol S Wijelath,3 Sara Cabbage,1 Jerry Ricks,1 Florian Bea,2 Morayma Reyes,1 Joanne van Ryn,4 Michael E Rosenfeld1,5 1Department of Pathology, University of Washington, Seattle, WA, USA; 2Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany; 3Department of Surgery, University of Washington, Seattle, WA, USA; 4Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany; 5Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, USA Objective: Thrombin has multiple proatherogenic effects including platelet activation and the induction of inflammatory processes. Recently, the cytokine oncostatin M has been shown to have proinflammatory effects. This study was designed to investigate the effects of thrombin inhibition on the initiation and progression of atherosclerosis and on the expression of oncostatin M. Methods: Apolipoprotein E-deficient mice at different ages were fed the thrombin inhibitor dabigatran etexilate. The mean lesion area was measured in the aortic sinus and in the innominate artery. CD45-positive cells within the aortic tissue were measured by flow cytometry. Oncostatin M expression was measured in the tissue sections by immunocytochemistry. Results: Treatment with dabigatran etexilate resulted in a significant reduction of the mean area of atherosclerotic lesions in the aortic sinus in both the young mice (11,176±1,500 µm2 (control) versus 3,822±836 µm2 (dabigatran etexilate), P

Atherosclerosis is a chronic disease hallmarked by chronic inflammation, endothelial dysfunction and lipid accumulation in the vasculature. Although lipid modification and deposition are thought to be a major source of the continuous inflammatory stimulus, a large body of evidence suggests that infectious agents may contribute to atherosclerotic processes. This could occur by either direct effects through infection of vascular cells and/or through indirect effects by induction of cytokine and acute phase reactant proteins by infection at other sites. Multiple bacterial and viral pathogens have been associated with atherosclerosis by seroepidemiological studies, identification of the infectious agent in human atherosclerotic tissue, and experimental studies demonstrating an acceleration of atherosclerosis following infection in animal models of atherosclerosis. This review will focus on those infectious agents for which biological plausibility has been demonstrated in animal models and on the challenges of proving a role of infection in human atherosclerotic disease.

Air Pollution has been associated with significant adverse health effects leading to increased morbidity and mortality. Cumulative epidemiological and experimental data have shown that exposure to air pollutants leads to increased cardiovascular ischemic events and enhanced atherosclerosis. The strongest associations are with the particulate matter (PM) components of air pollution, and it appears that the smaller particles are more pathogenic and have a greater ability to induce systemic vascular effects. Although it is unclear how the exposure to air pollutants can affect the systemic vasculature, it is likely that various mechanistic pathways participate including the induction of systemic pro-oxidant and pro-inflammatory responses which are now thought to play a central role. Air pollutants lead to oxidative alterations of plasma lipoproteins that increase the atherogenicity of low-density lipoproteins (LDL) and decrease the protective effects of high-density lipoproteins (HDL). This chapter reviews the epidemiological, clinical and experimental animal evidence that support the association of air pollutants with systemic pro-oxidant effects resulting in alteration of plasma lipoproteins and enhanced atherosclerosis.

Objective— Inflammatory mechanisms are involved in atherosclerotic plaque rupture and subsequent thrombin formation. Thrombin not only plays a central role in thrombus formation and platelet activation, but also in the induction of inflammatory processes. We assessed the hypothesis that melagatran, a direct thrombin inhibitor, attenuates plaque progression and promotes stability of advanced atherosclerotic lesions. Methods and Results— Melagatran (500 μmol/kg/d) or control diet was administered to apolipoprotein E–deficient mice (n=54) with advanced atherosclerotic lesions. Treatment reduced lesion progression in brachiocephalic arteries ( P P P 2 versus 126 819±51730 μm 2 ; P P P P Conclusions— The direct thrombin inhibitor melagatran reduces lesion size and may promote plaque stability in apolipoprotein E–deficient mice, possibly through reduced activation of proinflammatory transcription factors and reduced synthesis of MMP-9.

Very low-density lipoprotein (VLDL) and LDL plasma levels are associated with cardiovascular mortality. Whereas VLDL/LDL lowering causes regression of early atherosclerotic lesions, less is known about the effects of aggressive lipid lowering on regression of advanced complex lesions. We therefore investigated the effect of VLDL/LDL lowering on pre-existing lesions in LDL receptor-deficient mice. Mice fed a high-fat diet for 16 weeks developed advanced lesions with fibrous caps, necrotic cores, and cholesterol clefts in the brachiocephalic artery. After an additional 14 weeks on a low-fat diet, plasma cholesterol levels decreased from 21.0 +/- 2.6 to 8.4 +/- 0.6 mmol/L, but lesions did not regress. Levels of VLDL/LDL were further lowered by using a helper-dependent adenovirus encoding the VLDL receptor (HD-Ad-VLDLR) under control of a liver-selective promoter. Treatment with HD-Ad-VLDLR together with a low-fat diet regimen resulted in reduced lesion size (cross-sectional area decreased from 146,272 +/- 19,359 to 91,557 +/- 15,738 microm2) and an 89% reduction in the cross-sectional lesion area occupied by macrophages compared to controls. These results show that aggressive VLDL/LDL lowering achieved by hepatic overexpression of VLDLR combined with a low-fat diet regimen induces regression of advanced plaques in the brachiocephalic artery of LDL receptor-deficient mice.

Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5′-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.

Objective— Advanced atherosclerotic lesions in the innominate arteries of chow-fed apolipoprotein E–deficient mice become highly calcified with 100% frequency by 75 weeks of age. The time course, cell types, and mechanism(s) associated with calcification were investigated. Methods and Results— The deposition of hydroxyapatite is preceded by the formation of fibro-fatty nodules that are populated by cells that morphologically resemble chondrocytes. These cells are spatially associated with small deposits of hydroxyapatite in animals between 45 and 60 weeks of age. Immunocytochemical analyses with antibodies recognizing known chondrocyte proteins show that these cells express the same proteins as chondrocytes within developing bone. Histological and electron microscopic analyses of lesions from animals between 45 and 60 weeks of age show that the chondrocyte-like cells are surrounded by dense connective tissue that stains positive for type II collagen. Nanocrystals of hydroxyapatite can be seen within matrix vesicles derived from the chondrocyte-like cells. In mice between 75 and 104 weeks of age, the lesions have significantly reduced cellularity and contain large calcium deposits. The few remaining chondrocyte-like cells are located adjacent to or within the large areas of calcification. Conclusions— Calcification of advanced lesions in chow-fed apolipoprotein E–deficient mice occurs reproducibly in mice between 45 and 75 weeks of age. The deposition of hydroxyapatite is mediated by chondrocytes, which suggests that the mechanism of calcification may in part recapitulate the process of endochondral bone formation.

Objective The present study describes the distribution of atherosclerotic lesions in the coronary arteries of chow-fed 60-week-old male ApoE−/−, 17-β-estradiol-treated ApoE−/−, and wild-type mice. Methods and results The histologic examination of coronary arteries in 12 ApoE−/− and 6 wild-type mice, in contrast to the distribution of atherosclerosis in human coronary arteries, reveals that the major lesions in the mouse are located in the valve sinus, including the origins of the coronary arteries. These retrovalvular lesions either stop abruptly at the orifice of the common coronary artery or extend a short distance onto the arterial trunks. The first segment and first branch of all the major coronary arteries, the usual sites of disease in humans, are protected from disease. Although the arterial trunks and the first level branches are free of disease, we found approximately four independent lesions per heart. Independent lesions are present in the heart in smaller, intramyocardial vessels. These lesions are comprised predominantly of macrophages and proteoglycan and exhibit little extracellular lipid. In some cases, the independent lesions occlude the lumen without evidence of myocardial infarct in the surrounding tissue. Conclusions The specificity of the localization of lesions in certain segments of the murine coronary tree suggests that fundamental properties found at different branch levels determine lesion location.

OBJECTIVES To evaluate whether antimicrobial chemotherapy prevents acceleration of atherosclerotic lesion development induced by infection with Chlamydia pneumoniae. METHODS ApoE-deficient mice which develop hyperlipidaemia and atherosclerosis spontaneously were inoculated intranasally with C. pneumoniae. Animals were treated with azithromycin for 6 weeks after the third inoculation and the atherosclerotic lesion areas in the aortic sinus were measured by computer-assisted morphometry. RESULTS At 12 weeks post-infection, infected untreated animals developed significantly larger lesion areas compared with sham-inoculated controls (8.7 x 10(4)+/-2.3 x 10(4) microm(2) versus 5.6 x 10(4)+/-2.4 x 10(4) microm(2)). However, there were no differences in lesion size of infected mice treated with azithromycin in comparison with untreated infected controls (11.0 x 10(4)+/-3.0 x 10(4) microm(2) versus 8.7 x 10(4)+/-2.3 x 10(4) microm(2)). CONCLUSIONS Antibiotic treatment against C. pneumoniae has no beneficial effects on hyperlipidaemia-induced atherosclerosis accelerated by C. pneumoniae in a mouse model.

Introduction: Currently there are no clinically available means of noninvasively detecting early atherosclerotic disease because these lesions are characterized by an accumulation of extracellular lipid and foam cells, but a lack of significant wall thickening or architectural distortion. Objective: We hypothesize that a paramagnetically labeled low density lipoprotein (LDL) could serve as a functional probe to detect sites of abnormal lipid metabolism in the vessel wall that represent sites of early disease. Methods: Isolated LDL was first incubated with manganese–mesoporphyrin, a hydrophobic MR contrast agent (MnMeso). Size exclusion chromatography and absorption mass spectroscopy were performed on the resulting samples to prove that an association between the two occurred. Subsequently, foam cell cultures (n=7) were incubated (10–30 μg/ml for 48 h) with these labeled lipoproteins and the T1 relaxivity of centrifuged pellets of these cells was determined by using an inversion recovery sequence on a 1.5T scanner. These results were compared to control measurements made from foam cell cultures fed unlabeled lipoproteins (n=7). Results: Measured T1 relaxation times of the cells fed the MnMeso–LDL (443.3 ± 51.8 ms) was significantly different from the T1 relaxivity obtained from cells fed unlabeled lipoproteins (661.3 ± 60.9 ms). These findings indicate that the amount of contrast bound to the constructed lipoproteins is sufficient to produce measurable MR signal changes noninvasively. Conclusions: The study results support the feasibility of future in vivo MR experiments with labeled lipoproteins to assess lipoprotein kinetics in the vessel wall, which will hopefully provide a means of detecting early atherosclerotic disease.

Background: Inflammatory processes are an integral component of the initiation, progression, and destabilization of atherosclerotic lesions. Tumor necrosis factor-α (TNF-α) is considered a primary mediator of inflammatory processes. Methods and results: The role of TNF-α in plaque progression and plaque destabilization was investigated in the innominate arteries of older TNF-α receptor p55 deficient mice that were generated on a hyperlipidemic apolipoprotein E deficient background (p55−/− apoE−/−). There were no significant differences in levels of circulating cytokines, plaque progression, plaque composition or features of plaque destabilization in p55−/− apoE−/− compared to wild type (p55+/+ apoE−/−) mice. Conclusions: Progression and destabilization of advanced atherosclerotic lesions does not seem to be mediated via the TNF-α receptor p55.

Objective To determine whether progestins counteract the cardioprotective effects of estrogen. Design Controlled animal study. Setting Academic laboratory environment. Animal(s) Female apolipoprotein E–deficient mice. Intervention(s) Mice were randomly assigned to groups receiving a sham operation plus placebo pellet, bilateral gonadectomy plus placebo pellet, or gonadectomy plus one of nine combinations of estrogen/progestin SC pellets. Main outcome measure(s) Total plasma cholesterol, body weight, fat depot weight, uterine weight and size, and the cross-sectional area of fatty streaks in the aortic sinus were measured in each animal. Result(s) After 8 weeks of treatment, plasma cholesterol levels were significantly higher only in the ovariectomized and sham-operated animals that received placebo pellets. No differences in plasma cholesterol were observed relative to the type or amount of progestin administered. There was a reduction in fatty streaks in all of the hormone treatment groups as compared with both the ovariectomized and sham-operated animals that received placebo pellets. Conclusion(s) There were no significant differences in lesion area in response to estrogen alone or to estrogen plus the different types and doses of progestins.

It is well documented that hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. This study was designed to investigate whether some of the atherosclerotic effects ascribed to HHcy are mediated by oxidative stress and nuclear factor kappa B (NFkappaB) activation in peritoneal macrophages of C57BL/6 mice fed a high (2%) methionine/low (1 mg/kg) folate diet for 12 weeks. Plasma homocysteine concentrations in mice fed methionine averaged 49 mol/L after 12 weeks of feeding, five times higher than that of controls. HHcy induced by methionine feeding significantly elevated oxidative stress, as measured by superoxide anion radical level (P.05) in peritoneal macrophages. Furthermore, NFkappaB binding activities of peritoneal macrophages were higher in the methionine group than in the control group. These results suggest that HHcy induced by methionine may intensify disturbances in peroxidation and inflammatory mediator activation in peritoneal macrophages, and is a possible mechanism of its atherogenic effects.

Recent studies suggest that the beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) in reducing cardiovascular events may in part, be independent of their capacity to lower plasma lipids. To test this hypothesis, simvastatin (50 mg/kg/d) was administered to 30-week-old apolipoprotein E deficient mice (apo E-/-) for 12, 18 and 24 weeks. In contrast to other experimental models and humans, simvastatin treatment increases plasma cholesterol levels in apo E-/- mice. Quantitative real-time polymerase chain reaction was used to quantify expression of tissue factor (TF) and monocyte chemoattractant protein-1 (MCP-1) in the aorta of each mouse. Expression of TF was reduced to 34, 24, and 13% of control levels at 12, 18 and 24 weeks, respectively, of simvastatin administration. Advanced lesions in the innominate arteries of the simvastatin treated mice had reduced levels of TF, fewer macrophages and reduced expression of early growth response-1 (Egr-1). In vitro studies in mouse macrophages demonstrated decreased lipopolysaccharide induced binding of nuclear proteins to the Egr-1 consensus DNA sequence following pretreatment with simvastatin. RNA levels for MCP-1 were reduced to 30% of control values following 24 weeks of simvastatin treatment. In conclusion, these data suggest that chronic administration of simvastatin to older apo E-/- mice can inhibit the expression of pro-thrombotic/pro-inflammatory genes within established atherosclerotic lesions via mechanisms that are independent of reductions in plasma lipids.

The uptake of oxidized low-density lipoproteins (oxLDL) by macrophages leading to conversion into foam cells is a seminal event in atherogenesis. Excessive accumulation of oxLDL can cause oxidative stress in foam cells leading to cell death and the progression and destabilization of atherosclerotic lesions. Oxidative stress induces a protective compensatory increase in the synthesis of the endogenous antioxidant glutathione (GSH). Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in GSH synthesis and is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM), which are products of separate genes. Treatment of RAW 264.7 mouse macrophages and mouse peritoneal macrophages with oxLDL (30 μg/mL) induces increased expression of bothGclcandGclmin vitro. The increase in mRNA occurs in part via increased transcription as demonstrated with luciferase reporter constructs. The promoters for bothGCLCandGCLMcontain consensus antioxidant response elements (AREs). Electrophoretic mobility shift assays revealed induction of nuclear factor binding to these AREs after treatment of RAW 264.7 cells and mouse peritoneal macrophages with oxLDL. Nuclear factor binding to the AREs is diminished by a single base pair substitution in the core sequence. Site-directed mutagenesis of the AREs within theGclcandGclmpromoters resulted in a decrease of oxLDL-induced luciferase activity. Supershift analyses revealed that oxLDL stimulates binding of the transcription factors Nrf1, Nrf2, and c-jun to the AREs. These data suggest that AREs play a direct role in mediating the induction of GSH synthesis by oxLDL and in protecting macrophages against oxidized lipid-induced oxidative stress.

Recent studies have suggested that infection withChlamydia pneumoniae (C pneumoniae)may contribute to the instability of atherosclerotic plaques and thrombosis and is associated with acute coronary events. Tissue factor (TF), a potent prothrombotic molecule, is expressed by macrophages and other cell types within atherosclerotic lesions and plays an essential role in thrombus formation after plaque rupture. Therefore the effects ofC pneumoniaeon induction of TF expression in macrophages were investigated. Infection of RAW mouse macrophages withC pneumoniaeinduced a time-dependent increase in procoagulant activity, expression of TF protein, and TF mRNA.C pneumoniaeinfection stimulated increased binding of nuclear proteins to the consensus DNA sequence for Egr-1, a key response element within the TF promoter, and increased the expression of Egr-1 protein. Transient transfections of RAW cells with mutated TF promoter constructs showed that the Egr-1 binding region is an important transcriptional regulator ofC pneumoniae–induced TF expression. Furthermore,C pneumoniae–stimulated phosphorylation of ERK1/2 and Elk-1 and pharmacological inhibition of mitogen-activated protein kinase activity reduced the expression of TF and Egr-1. Antibody and polymyxin B blocking of the Toll-like receptor 4 (TLR4) partially reduced theC pneumoniae–induced expression of TF and Egr-1. In conclusion, theC pneumoniae–induced increase in TF expression in macrophages is mediated in part by Egr-1, signaling through TLR4, and activation of the MEK-ERK1/2 pathway.

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-α in foam cells despite inhibition of nuclear factor-κB 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.

Studies linking cholesterol levels to the development of colorectal neoplasia are inconsistent, and Mendelian randomization has been suggested as a way to help avoid problems with confounding and reverse causation.We genotyped individuals who received a colonoscopy at Group Health (1998-2007) for 96 of 102 single-nucleotide polymorphisms identified by the Global Lipids Genetics Consortium. Participants included 139 advanced adenoma cases, 518 non-advanced adenoma cases, 380 non-adenomatous polyp cases, and 754 polyp-free controls. All had at least one available pre-colonoscopy lipid measurement from electronic records maintained by Group Health.Advanced adenoma cases were more likely than controls to have higher pre-colonoscopy zenith low-density lipoprotein (LDL), triglycerides (TG), and total cholesterol (TC) (odds ratio, OR per 20 mg/dL LDL increase: 1.16, 95 % confidence interval, CI 1.03-1.30; per 40 mg/dL TG increase: 1.09, 1.03-1.16; and per 20 mg/dL TC increase: 1.09, 1.02-1.18). For these traits, genotype-polyp ORs using weighted allele scores were not statistically significant (OR per increase in score scaled to a 20 mg/dL LDL increase: 1.17, 0.78-1.75; a 40 mg/dL TG increase: 1.12, 0.91-1.38; a 20 mg/dL TC increase: 0.99, 0.71-1.38).Cholesterol levels may be associated with advanced adenomas, but larger studies are warranted to determine whether this association can be attributed to genetics.