Diesel Exhaust Induces Mitochondrial Dysfunction, Hyperlipidemia, and Liver Steatosis.

OBJECTIVE: Air pollution is associated with increased cardiovascular morbidity and mortality, as well as dyslipidemia and metabolic syndrome. Our goal was to dissect the mechanisms involved. Approach and Results: 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 (9-hydroxyoctadecadienoic) and 13-HODE (13-hydroxyoctadecadienoic), lipid, and carbohydrate metabolism. Findings were corroborated, and mechanisms were further assessed in HepG2 hepatocytes in culture. ApoE knockout mice exposed to inhaled diesel exhaust (DE, 6 h/d, 5 days/wk for 16 weeks) exhibited elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and higher hepatic levels of 9-HODE and 13-HODE, as compared to control mice exposed to filtered air. A direct effect of DE 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 increased triglyceride content and significant downregulation of ACAD9 mRNA expression. Treatment of HepG2 cells with DE particles and oleic acid did not alter de novo lipogenesis but inhibited total, mitochondrial, and ATP-linked oxygen consumption rate, indicative of mitochondrial dysfunction. Treatment of isolated mitochondria, prepared from mouse liver, with DE particles and oleic acid also inhibited mitochondrial complex activity and ß-oxidation. CONCLUSIONS: DE exposure leads to dyslipidemia and liver steatosis in ApoE knockout mice, likely due to mitochondrial dysfunction and decreased lipid catabolism.

Increased calcification in osteoprotegerin-deficient smooth muscle cells: Dependence on receptor activator of NF-κB ligand and interleukin 6.

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.

Diesel exhaust induces systemic lipid peroxidation and development of dysfunctional pro-oxidant and pro-inflammatory high-density lipoprotein.

OBJECTIVE: To evaluate whether exposure to air pollutants induces oxidative modifications of plasma lipoproteins, resulting in alteration of the protective capacities of high-density lipoproteins (HDLs). APPROACH AND RESULTS: We exposed apolipoprotein E-deficient mice to diesel exhaust (DE) at ≈ 250 µg/m(3) for 2 weeks, filtered air (FA) for 2 weeks, or DE for 2 weeks, followed by FA for 1 week (DE+FA). DE led to enhanced lipid peroxidation in the brochoalveolar lavage fluid that was accompanied by effects on HDL functionality. HDL antioxidant capacity was assessed by an assay that evaluated the ability of HDL to inhibit low-density lipoprotein oxidation estimated by 2',7'-dichlorofluorescein fluorescence. HDL from DE-exposed mice exhibited 23,053 ± 2844 relative fluorescence units, higher than FA-exposed mice (10,282 ± 1135 relative fluorescence units, P<0.001) but similar to the HDL from DE+FA-exposed mice (22,448 ± 3115 relative fluorescence units). DE effects on HDL antioxidant capacity were negatively correlated with paraoxonase enzymatic activity, but positively correlated with levels of plasma 8-isoprostanes, 12-hydroxyeicosatetraenoic acid, 13-hydroxyoctadecadienoic acid, liver malondialdehyde, and accompanied by perturbed HDL anti-inflammatory capacity and activation of the 5-lipoxygenase pathway in the liver. CONCLUSIONS: DE emissions induced systemic pro-oxidant effects that led to the development of dysfunctional HDL. This may be one of the mechanisms by which air pollution contributes to enhanced atherosclerosis.

Progression of experimental lesions of atherosclerosis: assessment by kinetic modeling of black-blood dynamic contrast-enhanced MRI.

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.

In Utero Exposure of Hyperlipidemic Mice to Diesel Exhaust: Lack of Effects on Atherosclerosis in Adult Offspring Fed a Regular Chow Diet.

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.

Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoE-deficient mice: potential role of chondrocyte-like cells.

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.

Dabigatran etexilate retards the initiation and progression of atherosclerotic lesions and inhibits the expression of oncostatin M in apolipoprotein E-deficient mice.

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 µm(2) (control) versus 3,822±836 µm(2) (dabigatran etexilate), P<0.05) and selectively in the older mice at 28 weeks (234,099±13,500 µm(2) (control) versus 175,226±16,132 µm(2) (dabigatran etexilate), P<0.05). There were also fewer CD45-positive cells within the aortas of the dabigatran-treated mice and enhanced NO production in endothelial cells pretreated with dabigatran. In addition, the expression of oncostatin M was reduced in the lesions of dabigatran etexilate-treated mice. CONCLUSION: Inhibition of thrombin by dabigatran retards the development of early lesions and the progression of some established lesions in ApoE-/- mice. It improves endothelial function and retards macrophage accumulation within the vascular wall. Dabigatran also inhibits the expression of oncostatin M, and this suggests that oncostatin M may play a role in the initiation and progression of atherosclerosis.

Chlamydia pneumoniae infection of lungs and macrophages indirectly stimulates the phenotypic conversion of smooth muscle cells and mesenchymal stem cells: potential roles in vascular calcification and fibrosis.

Two hallmarks of advanced atherosclerosis are calcification and fibrosis. We hypothesized that Chlamydia pneumoniae infection may contribute to atherosclerosis by inducing the conversion of vascular smooth muscle cells to calcifying cells or by converting mesenchymal stem cells to osteochondrocytic or fibroblastic phenotypes. In this study, direct infection of bovine aortic smooth muscle cells (BSMCs) did not induce the expression of alkaline phosphatase or the deposition of extracellular calcium phosphate. However, conditioned media from C. pneumoniae-infected macrophages accelerated conversion of BSMCs to a calcifying phenotype. Treatment of the conditioned media with an anti-TNF-alpha blocking antibody abrogated this stimulatory effect. Treatment of perivascular Sca-1+, CD31-, CD45- cells from apoE-/- mouse aortas with the conditioned media from infected macrophages induced the Sca-1+ cells to produce collagen II, an additional marker of an osteochondrocytic phenotype. Treatment of mouse coronary perivascular Sca-1+, CD31-, CD45- cells with the supernatant from homogenates of C. pneumoniae-infected mouse lungs as compared to noninfected lungs induced expression of the Collagen 1α1 gene and deposition of collagen. Therefore, an increase in plasma cytokines or other factors in response to respiratory infection with C. pneumoniae or infection of macrophages within the blood vessel could contribute to both calcification and fibrosis of advanced atherosclerotic lesions.

The acute phase reactant response to respiratory infection with Chlamydia pneumoniae: implications for the pathogenesis of atherosclerosis.

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.