Atherogenesis In Mice Research Articles

Abstract 18885: CTRP1, an Adipokine Causing Endothelial Dysfunction Through Upregulation of Arginase 1, is Associated With the Severity of Coronary Artery Disease in Patients and Promotes Atherogenesis in Mice

Background: C1q/TNF-related protein (CTRP) 1 was initially identified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins. However, vascular functions of CTRP1 remain largely undetermined. Here, we sought to investigate whether CTRP1 influences the pathogenesis of atherosclerosis. Methods and Results: We analyzed CTRP1 levels in sera of coronary artery disease (CAD) patients (n=451) and non-CAD controls (n=686), in coronary endarterectomy specimens (n=26) and non-atherosclerotic internal mammary arteries (n=32), and in peripheral blood mononuclear cells (PBMCs) from severe CAD patients (n=58) and non-CAD controls (n=25). CTRP1 levels were higher in sera, endarterectomy specimens and PBMCs from CAD patients compared with controls, and were related to CAD severity. Immunohistochemistry of human femoral arteries exhibited VE-cadherin loci-associated increased CTRP1 expression in atherosclerotic plaques. In cultured endothelial cells, CTRP1 stimulation resulted in reduced nitric oxide (NO) bioavailability and promotion of inflammatory reactions (increased production of inflammatory cytokines and induction of adhesion molecules). Intravital microscopy exhibited impaired endothelium-dependent arteriole dilation and enhanced leukocyte-endothelium interactions after injection of recombinant CTRP1, whereas improved arteriole dilation and decreased leukocyte-endothelium interactions were observed in CTRP1 knockout mice. Furthermore, we found that arginase 1 was significantly increased by CTRP1 in a dose-dependent fashion, thereby leading to eNOS-uncoupling and reactive oxygen species generation. Inhibition of arginase activity by synthetic chemicals markedly improved CTRP1-dependent endothelial dysfunction. Finally, intraperitoneal injection of recombinant CTRP1 promoted atherogenesis in apoE-/- mice. In contrast, proatherogenic effects were significantly attenuated in CTRP1-/-/apoE-/- double knockout mice. Conclusions: Our findings have demonstrated that CTRP1 is a pro-atherogenic adipokine, likely by modulating arginase 1 expression and enzyme activity. CTPP1 may represent a crucial therapeutic target in atherosclerosis.

NG2 Proteoglycan Ablation Reduces Foam Cell Formation and Atherogenesis via Decreased Low-Density Lipoprotein Retention by Synthetic Smooth Muscle Cells.

Obesity and hyperlipidemia are critical risk factors for atherosclerosis. Because ablation of NG2 proteoglycan in mice leads to hyperlipidemia and obesity, we investigated the impact of NG2 ablation on atherosclerosis in apoE null mice. Immunostaining indicates that NG2 expression in plaque, primarily by synthetic smooth muscle cells, increases during atherogenesis. NG2 ablation unexpectedly results in decreased (30%) plaque development, despite aggravated obesity and hyperlipidemia. Mechanistic studies reveal that NG2-positive plaque synthetic smooth muscle cells in culture can sequester low-density lipoprotein to enhance foam-cell formation, processes in which NG2 itself plays direct roles. In agreement with these observations, low-density lipoprotein retention and lipid accumulation in the NG2/ApoE knockout aorta is 30% less than that seen in the control aorta. These results indicate that synthetic smooth muscle cell-dependent low-density lipoprotein retention and foam cell formation outweigh obesity and hyperlipidemia in promoting mouse atherogenesis. Our study sheds new light on the role of synthetic smooth muscle cells during atherogenesis. Blocking plaque NG2 or altering synthetic smooth muscle cells function may be promising therapeutic strategies for atherosclerosis.

Food supplementation with rice bran enzymatic extract prevents vascular apoptosis and atherogenesis in ApoE-/- mice.

Atherosclerosis is associated with reduced mononuclear cell (MNC) telomere length, and senescent cells have been detected in atherosclerotic plaques. Rice bran is a source of γ-oryzanol, phytosterols and tocols with potential lipid-lowering, antioxidant and anti-inflammatory activities. Here, we tested the hypothesis that rice bran enzymatic extract (RBEE) impacts on apoptosis, telomere length and atherogenesis in mice. Seven-week-old male ApoE-/- mice were fed high-fat diet (HFD) or isocaloric HFD supplemented with 5% (w/w) RBEE for 23weeks. Wild-type mice of the same age were kept under standard diet as controls. RBEE treatment reduced total cholesterol (19.24±1.63 vs 24.49±1.71mmol/L) and triglycerides (1.13±0.18 vs 1.75±0.22mmol/L) and augmented HDL-cholesterol (1.86±0.20 vs 1.07±0.20mmol/L). RBEE attenuated macrophage infiltration by 56.69±4.65% and plaque development (7737±836 vs 12,040±1001μm2) in the aortic sinus. In the aorta, RBEE treatment reduced expression of the apoptosis pathway components p16, p53 and bax/bcl-2 ratio. RBEE prevented apoptosis of aortic endothelial cells (2.81±0.71-1.14±0.35 apoptotic nuclei/ring for ApoE-/- HFD and ApoE-/- HFD 5% RBEE, respectively). In contrast, MNC of RBEE-fed mice exhibited enhanced apoptosis marker expression with increased p53 and bax/bcl-2 protein levels. Compared to WT, ApoE-/- mice on HFD were characterized by significant telomere shortening in aorta (11±2%) and MNC (73±7%), which was reduced by supplementation with RBEE (aorta: 40±7%; MNC: 105±10%). Expression of telomere repeat-binding factor 2 was increased in RBEE-fed mice. Long-term food supplementation with RBEE lowers cholesterol and prevents atherosclerotic plaque development in ApoE-/- mice. Differential regulation of vascular and MNC apoptosis and senescence were identified as potential mechanisms.

Aggravated restenosis and atherogenesis in ApoCIII transgenic mice but lack of protection in ApoCIII knockouts: the effect of authentic triglyceride-rich lipoproteins with and without ApoCIII

Previously, our group and others have demonstrated a causative relationship between severe hypertriglyceridaemia and atherogenesis in mice. Furthermore, clinical investigations have shown high levels of plasma Apolipoprotein C-III (ApoCIII) associated with hypertriglyceridaemia and even cardiovascular disease. However, it remains unclear whether ApoCIII affects restenosis in vivo, and whether such an effect is mediated by ApoCIII alone, or in combination with hypertriglyceridaemia. We sought to investigate ApoCIII in restenosis and clarify how smooth muscle cells (SMCs) respond to authentic triglyceride-rich lipoproteins (TRLs) with or without ApoCIII (TRLs ± ApoCIII). ApoCIII transgenic (ApoCIIItg) and knockout (ApoCIII-/-) mice underwent endothelial denudation to model restenosis. Here, ApoCIIItg mice displayed severe hypertriglyceridaemia and increased neointimal formation compared with wild-type (WT) or ApoCIII-/- mice. Furthermore, increased proliferating cell nuclear antigen (PCNA)-positive cells, Mac-3, and vascular cell adhesion protein-1 (VCAM-1) expression, and 4-hydroxynonenal (4HNE) production were found in lesion sites. ApoCIIItg and ApoCIII-/- mice were then crossed to low-density lipoprotein receptor-deficient (Ldlr-/-) mice and fed an atherogenic diet. ApoCIIItg/Ldlr-/- mice had significantly increased atherosclerotic lesions. However, there was no statistical difference in restenosis between ApoCIII-/- and WT mice, and in atherosclerosis between ApoCIII/Ldlr double knockout and Ldlr-/- mice. SMCs were then incubated in vitro with authentic TRLs ± ApoCIII isolated from extreme hypertriglyceridaemia glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1-deficient (GPIHBP1-/-) mice crossed with ApoCIIItg or ApoCIII-/- mice. It was shown that TRLs + ApoCIII promoted SMC proliferation, VCAM-1 expression, and reactive oxygen species (ROS) production, and activated the Akt pathway. Scavenging ROS significantly reduced SMC activation caused by TRLs + ApoCIII. Severe hypertriglyceridaemia resulting from ApoCIII overexpression promotes restenosis and atherosclerosis. Furthermore, we demonstrated that TRLs + ApoCIII promotes SMC proliferation.

TLR4 antagonist attenuates atherogenesis in LDL receptor-deficient mice with diet-induced type 2 diabetes

Although a large number of studies have well documented a key role of toll-like receptor (TLR)4 in atherosclerosis, it remains undetermined if TLR4 antagonist attenuates atherogenesis in mouse model for type 2 diabetes. In this study, we induced type 2 diabetes in low-density lipoprotein receptor-deficient (LDLR−/−) mice by high-fat diet (HFD). At 8 weeks old, 20 mice were fed HFD and 20 mice fed regular chow (RC) for 24 weeks. In the last 10 weeks, half HFD-fed mice and half RC-fed mice were treated with Rhodobacter sphaeroides lipopolysaccharide (Rs-LPS), an established TLR4 antagonist. After the treatment, atherosclerotic lesions in aortas were analyzed. Results showed that the HFD significantly increased bodyweight, glucose, lipids including total cholesterol, triglycerides and free fatty acids, and insulin resistance, indicating that the HFD induced type 2 diabetes in LDLR−/− mice. Results also showed that Rs-LPS had no effect on HFD-increased metabolic parameters in both nondiabetic and diabetic mice. Lipid staining of aortas and histological analysis of cross-sections of aortic roots showed that diabetes increased atherosclerotic lesions, but Rs-LPS attenuated atherogenesis in diabetic mice. Furthermore, immunohistochemical studies showed that Rs-LPS reduced infiltration of monocytes/macrophages and expression of interleukin (IL)-6 and matrix metalloproteinase-9 in atherosclerotic lesions of diabetic mice. Finally, the antagonistic effect of Rs-LPS on TLR4 was demonstrated by our in vitro studies showing that Rs-LPS inhibited IL-6 secretion from macrophages and endothelial cells stimulated by LPS or LPS plus saturated fatty acid palmitate. Taken together, our study demonstrated that TLR4 antagonist was capable of attenuating vascular inflammation and atherogenesis in mice with HFD-induced type 2 diabetes.

Treatment with KLEPTOSE® CRYSMEB reduces mouse atherogenesis by impacting on lipid profile and Th1 lymphocyte response

The ability of pharmacological agents to target both “classical” risk factors and inflammation may be key for successful outcomes in the prevention and treatment of atherogenesis. Among the promising drugs interfering with cholesterol metabolism, we investigated whether methyl beta-cyclodextrin (KLEPTOSE® CRYSMEB) could positively impact on atherogenesis, lipid profile and atherosclerotic plaque inflammation in ApoE−/− mice. Eleven-week old ApoE−/− mice were fed either a normal diet (N.D.) or a high-cholesterol diet (H.D.), resulting in different levels of hypercholesterolemia. KLEPTOSE® CRYSMEB (40mg/kg) or vehicle was intraperitoneally administrated 3 times per week in the last 16weeks before euthanasia in mice under N.D. and in the last 11weeks under H.D. Treatment with KLEPTOSE® CRYSMEB reduced triglyceride serum levels in both atherogenesis mouse models. In H.D. mice, treatment with KLEPTOSE® CRYSMEB increased HDL-cholesterol levels and reduced free fatty acids and spleen weight. In both mouse models, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size in thoraco-abdominal aortas and intraplaque T lymphocyte content, but did not induce relevant improvements in other histological parameters of vulnerability (macrophage, neutrophil, MMP-9 and collagen content). Conversely and more markedly in H.D. mice, treatment with KLEPTOSE® CRYSMEB was associated with a reduction in genetic markers of Th1-mediated immune response. In vitro, KLEPTOSE® CRYSMEB dose-dependently abrogated Th1 proliferation and IFNγ release. In conclusion, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size by improving triglyceride serum levels and Th1-mediated response. These results indicate this drug as a potential tool for blocking atheroprogression associated with different severity degrees of hypercholesterolemia.

Involvement of Mitochondrial (mt) DNA Oxidative Damage and Circulating Fragments in Early Stages of Atherogenesis

Mitochondrial dysfunction underlies many processes in the development of atherosclerosis, including generation of reactive oxygen species, apoptosis, and inflammation. Oxidative mtDNA damage is a proximate cause of mitochondrial dysfunction and there is growing evidence that it can directly promote atherosclerosis. The mechanism of this action remains unclear, but may include initiation of apoptosis and inflammation by mtDNA oxidation. Previously, we have demonstrated protective role of mitochondria‐targeted DNA glycosylase Ogg1 against oxidative stress and apoptosis in cell culture and against vascular injury in animal models. In the present study we tested the hypothesis that transgenic modulation of Ogg1 coordinately regulates atherogenesis in mice fed a high fat diet. Wild type (WT) mice, Ogg1 knock‐out (KO) mice and KO mice transgenically overexpressing mitochondria‐targeted Ogg1 (KO‐Tg) were fed Western diet and analyzed for mtDNA damage and signs of atherogenesis. When compared to WT animals, KO mice displayed significantly higher mtDNA oxidative damage in cardiac tissue, elevated cell apoptosis and increased size of fatty streaks in aortic valve. These effects were eliminated in KO‐Tg mice. Interestingly, mtDNA oxidative damage correlated with the abundance of plasma pro‐inflammatory mtDNA fragments. These results indicate that enhanced mtDNA repair suppresses apoptosis and atherogenesis, suggesting that mtDNA and its integrity could be an important target for atheroprotection. Supported by NIH.

The inhibition of macrophage foam cell formation by 9-cis β-carotene is driven by BCMO1 activity.

Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15’-monooxygenase (BCMO1) is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis.

Lymphocytes in atherosclerosis.

Atherosclerosis is the major cause of cardiovascular disease, and cardiovascular disease remains the primary cause of death in the United States. The primary complication for patients with type 2 diabetes mellitus (T2D) is cardiovascular disease.1–8 Because the incidences of T2D and obesity are drastically increasing in the United States and now worldwide, understanding how atherosclerosis is accelerated in these diseases becomes even more important to discern. Although many scientific advances in research on atherosclerosis have been made by multiple groups during the past 30 years, particularly about the function of endothelial and smooth muscle biology and macrophage foam cell formation in atherosclerotic plaque development, the roles of immune cells in regulating atherosclerosis onset and progression are still being elucidated. It was widely thought that immune cells played little part in atherogenesis until 1986, when Hansson et al9–12 reported the presence of lymphocytes within atherosclerotic lesions; yet it was many years later until it was widely accepted that immune cells play an important role in atherogenesis. Since 1986, the viewpoint has changed significantly, but details on lymphocyte subsets and mechanisms are still emerging. Moreover, controversy still exists on the roles that some lymphocyte subsets play in atherosclerosis. These discrepancies in findings are at least partly because of differences in mouse models used, diet composition, and length of time on diet for atherosclerosis measurements. However, despite a few ongoing controversies, there are many studies on immune cells in atherogenesis in mice that show clear findings.10,12–23 For example, CD4+ T lymphocytes have been shown to accelerate atherosclerosis.18,24–29 Activated CD4 T cells can be distinguished based on their phenotype, and the predominant CD4+ effector T cells studied to date in the context of atherosclerosis are Th1, …

Vitamin A-deficient diet accelerated atherogenesis in apolipoprotein E(-/-) mice and dietary β-carotene prevents this consequence.

Vitamin A is involved in regulation of glucose concentrations, lipid metabolism, and inflammation, which are major risk factors for atherogenesis. However, the effect of vitamin A deficiency on atherogenesis has not been investigated. Therefore, the objective of the current study was to examine whether vitamin A deficiency accelerates atherogenesis in apolipoprotein E-deficient mice (apoE−/−). ApoE−/− mice were allocated into the following groups: control, fed vitamin A-containing chow diet; BC, fed chow diet fortified with Dunaliella powder containing βc isomers; VAD, fed vitamin A-deficient diet; and VAD-BC group, fed vitamin A-deficient diet fortified with a Dunaliella powder. Following 15 weeks of treatment, liver retinol concentration had decreased significantly in the VAD group to about 30% that of control group. Vitamin A-deficient diet significantly increased both plasma cholesterol concentrations and the atherosclerotic lesion area at the aortic sinus (+61%) compared to the control group. Dietary βc fortification inhibited the elevation in plasma cholesterol and retarded atherogenesis in mice fed the vitamin A-deficient diet. The results imply that dietary vitamin A deficiency should be examined as a risk factor for atherosclerosis and that dietary βc, as a sole source of retinoids, can compensate for vitamin A deficiency.

Abstract 15768: Mitochondria-Targeted DNA Repair Glycosylase Ogg1 Suppresses Early Stage of Atherogenesis in Ogg1 Deficient Mice

Introduction: Reactive oxygen species (ROS) play a key role in the development of atherosclerosis. Mitochondria are a main source of endogenous ROS in the cell. Mitochondrial DNA (mtDNA) is sensitive to oxidation and our previous results from cultured cell and intact animal models suggest that increasing mtDNA repair prevents both oxidative mtDNA damage and associated cytotoxicity and cellular dysfunction. Involvement of oxidative mtDNA damage in disorders characterized by chronic oxidative stress has been less thoroughly studied. Hypothesis: In the present study we tested the hypothesis that transgenic modulation of Ogg1, a DNA glycosylase mediating the first step in the base excision repair of oxidative mtDNA damage, coordinately regulates atherogenesis in mice fed a high fat diet. Methods: Wild type (WT) mice, Ogg1 knock-out (KO) mice and KO mice transgenically overexpressing mitochondria-targeted Ogg1 (KO-Tg) were fed pro-atherogenic Western type diet for 14 weeks and analyzed for mtDNA damage and signs of atherogenesis. Results: KO mice fed a high fat diet had increased oxidative mtDNA damage in cardiac tissue, whereas KO-Tg animals did not differ from WT mice (WT: 0.18 ± 0.04; KO: 0.35 ± 0.04; KO-Tg: 0.15 ± 0.01 lesions per 10 4 bp; n=3, P<0.05; quantitative Southern blot analysis). We did not observe significant atherosclerotic plaque formation in the aortic valve of animals from any group; however appearance of fatty streaks, indicative of early plaque development, was more evident in KO mice (WT: 179 ± 20; KO: 384 ± 59 pixels, n=4, P<0.05; immunohistochemistry for Fc receptor - general marker of inflammatory cells). This effect was completely blocked in KO-Tg mice. We found increased number of apoptotic cells in the aortic valve of KO, but not KO-Tg mice (WT: 2.00 ± 0.50; KO: 4.25 ± 0.48; KO-Tg: 2.14 ± 0.85 apoptotic cells, n=4, P<0.05; TUNEL assay). Conclusion: Our data demonstrate that Ogg1 deficiency in mice fed a high fat diet leads to increased oxidative mtDNA damage, appearance of fatty streaks and cell apoptosis. In contrast, enhancement of mtDNA repair with mitochondria-targeted Ogg1 reduces fatty streaks formation and apoptosis induced by a high fat diet. These results suggest mtDNA damage and repair could be important targets for atheroprotection.

Inputs for subject-specific computational fluid dynamics simulation of blood flow in the mouse aorta.

Mouse models are an important way for exploring relationships between blood hemodynamics and eventual plaque formation. We have developed a mouse model of aortic regurgitation (AR) that produces large changes in plaque burden with charges in hemodynamics [Zhou et al., 2010, "Aortic Regurgitation Dramatically Alters the Distribution of Atherosclerotic Lesions and Enhances Atherogenesis in Mice," Arterioscler. Thromb. Vasc. Biol., 30(6), pp. 1181-1188]. In this paper, we explore the amount of detail needed for realistic computational fluid dynamics (CFD) calculations in this experimental model. The CFD calculations use inputs based on experimental measurements from ultrasound (US), micro computed tomography (CT), and both anatomical magnetic resonance imaging (MRI) and phase contrast MRI (PC-MRI). The adequacy of five different levels of model complexity (a) subject-specific CT data from a single mouse; (b) subject-specific CT centerlines with radii from US; (c) same as (b) but with MRI derived centerlines; (d) average CT centerlines and averaged vessel radius and branching vessels; and (e) same as (d) but with averaged MRI centerlines) is evaluated by demonstrating their impact on relative residence time (RRT) outputs. The paper concludes by demonstrating the necessity of subject-specific geometry and recommends for inputs the use of CT or anatomical MRI for establishing the aortic centerlines, M-mode US for scaling the aortic diameters, and a combination of PC-MRI and Doppler US for estimating the spatial and temporal characteristics of the input wave forms.

Select Articles Published on the Topic of Coronary Heart Disease in 2013

Select Articles Published on the Topic of Coronary Heart Disease in 2013

Bone Marrow Deficiency of MCPIP1 Results in Severe Multi-Organ Inflammation but Diminishes Atherogenesis in Hyperlipidemic Mice

ObjectiveMCPIP1 is a newly identified protein that profoundly impacts immunity and inflammation. We aim to test if MCPIP1 deficiency in hematopoietic cells results in systemic inflammation and accelerates atherogenesis in mice.Approach and ResultsAfter lethally irradiated, LDLR−/− mice were transplanted with bone marrow cells from either wild-type or MCPIP1−/− mice. These chimeric mice were fed a western-type diet for 7 weeks. We found that bone marrow MCPIP1−/− mice displayed a phenotype similar to that of whole body MCPIP1−/− mice, with severe systemic and multi-organ inflammation. However, MCPIP1−/− bone marrow recipients developed >10-fold less atherosclerotic lesions in the proximal aorta than WT bone marrow recipients, and essentially no lesions in en face aorta. The diminishment in atherosclerosis in bone marrow MCPIP1−/− mice may be partially attributed to the slight decrease in their plasma lipids. Flow cytometric analysis of splenocytes showed that bone marrow MCPIP1−/− mice contained reduced numbers of T cells and B cells, but increased numbers of regulatory T cells, Th17 cells, CD11b+/Gr1+ cells and CD11b+/Ly6Clow cells. This overall anti-atherogenic leukocyte profile may also contribute to the reduced atherogenesis. We also examined the cholesterol efflux capability of MCPIP1 deficient macrophages, and found that MCPIP1deficiency increased cholesterol efflux to apoAI and HDL, due to increased protein levels of ABCA1 and ABCG1.ConclusionsHematopoietic deficiency of MCPIP1 resulted in severe systemic and multi-organ inflammation but paradoxically diminished atherogenesis in mice. The reduced atheroegensis may be explained by the decreased plasma cholesterol levels, the anti-atherogenic leukocyte profile, as well as enhanced cholesterol efflux capability. This study suggests that, while atherosclerosis is a chronic inflammatory disease, the mechanisms underlying atherogenesis-associated inflammation in arterial wall versus the inflammation in solid organs may be substantially different.

Impaired Cholesterol Metabolism and Enhanced Atherosclerosis in Clock Mutant Mice

Clock is a key transcription factor that positively controls circadian regulation. However, its role in plasma cholesterol homeostasis and atherosclerosis has not been studied. We show for the first time that dominant-negative Clock mutant protein (Clock(Δ19/Δ19)) enhances plasma cholesterol and atherosclerosis in 3 different mouse models. Detailed analyses revealed that Clk(Δ19/Δ19)Apoe(-/-) mice display hypercholesterolemia resulting from the accumulation of apolipoprotein B48-containing cholesteryl ester-rich lipoproteins. Physiological studies showed that enhanced cholesterol absorption by the intestine contributes to hypercholesterolemia. Molecular studies indicated that the expression of Niemann Pick C1 Like 1, Acyl-CoA:Cholesterol acyltransferase 1, and microsomal triglyceride transfer protein in the intestines of Clk(Δ19/Δ19)Apoe(-/-) mice was high and that enterocytes assembled and secreted more chylomicrons. Furthermore, we identified macrophage dysfunction as another potential cause of increased atherosclerosis in Clk(Δ19/Δ19)Apoe(-/-) mice. Macrophages from Clk(Δ19/Δ19)Apoe(-/-) mice expressed higher levels of scavenger receptors and took up more modified lipoproteins compared with Apoe(-/-) mice, but they expressed low levels of ATP binding casette protein family A member 1 and were defective in cholesterol efflux. Molecular studies revealed that Clock regulates ATP binding casette protein family A member 1 expression in macrophages by modulating upstream transcription factor 2 expression. Clock(Δ19/Δ19) protein enhances atherosclerosis by increasing intestinal cholesterol absorption, augmenting uptake of modified lipoproteins by macrophages, and reducing cholesterol efflux from macrophages. These studies establish that circadian Clock activity is crucial in maintaining low plasma cholesterol levels and in reducing atherogenesis in mice.

Increased Atherosclerotic Lesions in LDL Receptor Deficient Mice With Hematopoietic Nuclear Receptor Rev‐erbα Knock‐ Down

BackgroundNuclear receptor Rev‐erbα plays important roles in circadian clock timing, lipid metabolism, adipogenesis, and vascular inflammation. However, the role of Rev‐erbα in atherosclerotic lesion development has not been assessed in vivo.Methods and ResultsThe nuclear receptor Rev‐erbα was knocked down in mouse haematopoietic cells by means of shRNA‐lentiviral transduction, followed by bone marrow transplantation into LDL receptor knockout mice. The Rev‐erbα protein in peripheral macrophage was reduced by 70% as compared to control mice injected with nontargeting shRNA lentivirus‐transduced bone marrow. A significant increase in atherosclerotic lesions was observed around the aorta valves as well as upon en face aorta analysis of Rev‐erbα knock‐down bone marrow recipients (P<0.01) as compared to the control mice, while plasma cholesterol, phospholipid, and triacylglycerol levels were not affected. Overexpression of Rev‐erbα in bone marrow mononuclear cells decreased inflammatory M1 while increasing M2 macrophage markers, while Rev‐erbα knock down increased the macrophage inflammatory phenotype in vitro and in vivo. Furthermore, treatment of differentiating macrophages with the Rev‐erbα ligand heme promoted expression of antiinflammatory M2 markers.ConclusionsThese observations identify hematopoietic cell Rev‐erbα as a new modulator of atherogenesis in mice.

T Helper Cell Polarization in Healthy People: Implications for Cardiovascular Disease

Atherosclerosis is a chronic inflammatory disease characterized by T lymphocyte infiltration into the atherosclerotic plaque. Assessments of T cell subtypes have demonstrated a predominance of CD4(+) T helper (Th) cells, implicated Th1 and Th17 immunity in both human and mouse atherogenesis, and provided some evidence suggesting protective roles of Th2 and T regulatory cells. Observations that certain inbred mouse strains have an inherent T helper bias suggest a genetic predisposition toward developing a particular T helper phenotype. This review summarizes our current understanding of mechanisms of antigen processing for major histocompatibility complex molecules, describes the different T helper cell subsets and their roles in atherosclerosis, and discusses mechanisms of genetic predisposition toward Th1/Th2 bias in mice. We also present data from our laboratory demonstrating inherent Th1/Th2 phenotypes in apparently healthy human volunteers that are stable over time and discuss the potential implications for cardiovascular disease.

Prevention of TGFβ induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr−/− mice

Angiotensin II (angII) accelerates atherosclerosis, but the mechanisms are not fully understood. The aim of this study was to determine whether TGFβ is required for angII-induced atherosclerosis. Ldlr-null mice fed a normal chow diet were infused with angII or saline for 28 days. A single injection of TGFβ neutralizing antibody 1D11 (2 mg/kg) prevented angII-induction of TGFβ1 levels, and strikingly attenuated angII-induced accumulation of aortic biglycan content. To study atherosclerosis, mice were infused with angII or saline for 4 weeks, and then fed Western diet for a further 6 weeks. 1D11 had no effect on systolic blood pressure or plasma cholesterol; however, angII-infused mice that received 1D11 had reduced atherosclerotic lesion area by 30% (P < 0.05). Immunohistochemical analyses demonstrated that angII induced both lipid retention and accumulation of biglycan and perlecan which colocalized with apoB. 1D11 strikingly reduced the effect of angII on biglycan but not perlecan. 1D11 decreased total collagen content (P < 0.05) in the lesion area without changing plaque inflammation markers (CD68 and CD45). Thus, this study demonstrates that neutralization of TGFβ attenuated angII stimulation of biglycan accumulation and atherogenesis in mice, suggesting that TGFβ-mediated biglycan induction is one of the mechanisms underlying angII-promoted atherosclerosis.

Developmental exposure to second-hand smoke increases adult atherogenesis and alters mitochondrial DNA copy number and deletions in apoE(-/-) mice.

Cardiovascular disease is a major cause of morbidity and mortality in the United States. While many studies have focused upon the effects of adult second-hand smoke exposure on cardiovascular disease development, disease development occurs over decades and is likely influenced by childhood exposure. The impacts of in utero versus neonatal second-hand smoke exposure on adult atherosclerotic disease development are not known. The objective of the current study was to determine the effects of in utero versus neonatal exposure to a low dose (1 mg/m3 total suspended particulate) of second-hand smoke on adult atherosclerotic lesion development using the apolipoprotein E null mouse model. Consequently, apolipoprotein E null mice were exposed to either filtered air or second-hand smoke: (i) in utero from gestation days 1–19, or (ii) from birth until 3 weeks of age (neonatal). Subsequently, all animals were exposed to filtered air and sacrificed at 12–14 weeks of age. Oil red-O staining of whole aortas, measures of mitochondrial damage, and oxidative stress were performed. Results show that both in utero and neonatal second-hand smoke exposure significantly increased adult atherogenesis in mice compared to filtered air controls. These changes were associated with changes in aconitase and mitochondrial superoxide dismutase activities consistent with increased oxidative stress in the aorta, changes in mitochondrial DNA copy number and deletion levels. These studies show that in utero or neonatal exposure to second-hand smoke significantly influences adult atherosclerotic lesion development and results in significant alterations to the mitochondrion and its genome that may contribute to atherogenesis.

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.