Low-density Lipoprotein Receptor-deficient Mice Research Articles

Danhong Injection Inhibits the Development of Atherosclerosis in Both Apoe−/− and Ldlr−/− Mice

Danhong injection (DHI), a certificated Chinese medical product made from radix salviae miltiorrhizae and flos carthami, is prescribed to patients with coronary heart disease in China. To investigate if DHI can inhibit atherosclerosis, apolipoprotein E-deficient (Apoe⁻/⁻) or low-density lipoprotein receptor-deficient (Ldlr⁻/⁻) mice on high-fat diet were divided into 2 groups and received daily intraperitoneal injection of saline and DHI, respectively, for 16 or 20 weeks. After the treatment, mouse aortas were collected to determine lesions, expression of adenosine triphosphate-binding cassette transporter A1 and tumor necrosis factor-α (TNF-α), and macrophage accumulation. Additionally, serum lipid profiles and expression of hepatic HMG-CoA reductase messenger RNA and low-density lipoprotein receptor protein were determined. We observed that DHI inhibited lesions in both Apoe⁻/⁻ and Ldlr⁻/⁻ mice. Associated with the decreased lesions, the aortic adenosine triphosphate-binding cassette transporter A1 expression was increased, whereas the macrophage accumulation was decreased in male Apoe⁻/⁻ mice and both male and female Ldlr⁻/⁻ mice. Although DHI reduced HMG-CoA reductase messenger RNA expression in both female Apoe⁻/⁻ and Ldlr⁻/⁻ mice, it decreased low-density lipoprotein cholesterol levels only in female Apoe⁻/⁻ mice. In addition to attenuation of lipopolysaccharide-induced expression of TNF-α, IL-1β, IL-6 in macrophages, and human C-reactive protein in hepatocytes, respectively, at the transcriptional level in vitro, DHI also reduced TNF-α protein expression in aortic root of both Apoe⁻/⁻ and Ldlr⁻/⁻ mice, suggesting the importance of the anti-inflammatory properties of DHI in the inhibition of lesion development. Taken together, our study demonstrates that DHI inhibits atherosclerosis in both Apoe⁻/⁻ and Ldlr⁻/⁻ mice with various mechanisms, including anti-inflammation. The inhibition of atherosclerosis can be attributed to the cardioprotective properties of DHI.

Pharmacological activation of the melanocortin system limits plaque inflammation and ameliorates vascular dysfunction in atherosclerotic mice.

Melanocortin peptides have been shown to elicit anti-inflammatory actions and to promote vascular endothelial function by activating type 1 and 3 melanocortin receptors. Here, we addressed whether these favorable properties of melanocortins could reduce atherosclerotic plaque inflammation and improve vasoreactivity in atherosclerotic mice. Low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 were fed a high-fat diet for 8 or 16 weeks and treated with either vehicle or a stable melanocortin analog, melanotan II (MT-II, 0.3 mg/kg per day, 4 weeks). We determined plaque uptake of fluorine-18-labeled fluorodeoxyglucose as a surrogate marker for atherosclerotic plaque inflammation and vascular function of the aorta by ex vivo analyses. MT-II had no effect on body weight or composition, or plasma cholesterol levels in atherosclerotic mice. Without attenuating atherosclerotic lesion size or lesional macrophage accumulation, MT-II treatment reduced fluorine-18-labeled fluorodeoxyglucose uptake in the atherosclerotic plaques. Resident macrophages in the lesions of MT-II-treated mice were polarized toward the anti-inflammatory M2 phenotype. Systemic inflammation was also attenuated by MT-II intervention as evidenced by decreased plasma levels of proinflammatory cytokines. In terms of aortic vasoreactivity, MT-II-treated mice showed enhanced endothelium-dependent relaxations, as well as promotion of vascular sensitivity to nitric oxide-mediated vasodilation, which were markedly impaired in control mice after prolonged duration of diet exposure. The present study demonstrates that pharmacological activation of the melanocortin system has therapeutic benefits in pre-established atherosclerosis by limiting plaque inflammation and promoting vascular endothelial function, which may provide a novel therapeutic approach for atherosclerosis.

New Therapy via Targeting Androgen Receptor in Monocytes/Macrophages to Battle Atherosclerosis

The male sex has a higher risk to develop coronary artery diseases, including atherosclerosis. The androgen receptor (AR) is expressed in several atherosclerosis-associated cell types, including monocytes/macrophages, endothelial cells (ECs), and smooth muscle cells (SMCs), but its pathophysiological role in each cell type during the development of atherosclerotic lesions remains unclear. Using the Cre-loxP system, we selectively knocked out AR in these 3 cell types and the resultant AR knockout (ARKO) mice, monocyte/macrophage ARKO, EC-ARKO, and SMC-ARKO, were then crossed with the low-density lipoprotein receptor (LDLR) deficient (LDLR(-/-)) mice to develop monocyte/macrophage ARKO-LDLR(-/-), EC-ARKO-LDLR(-/-), and SMC-ARKO-LDLR(-/-) mice for the study of atherosclerosis. The results showed that the monocyte/macrophage ARKO-LDLR(-/-) mice had reduced atherosclerosis compared with the wild-type-LDLR(-/-) control mice. However, no significant difference was detected in EC-ARKO-LDLR(-/-) and SMC-ARKO-LDLR(-/-) mice compared with wild-type-LDLR(-/-) mice, suggesting that the AR in monocytes/macrophages, and not in ECs and SMCs, plays a major role to promote atherosclerosis. Molecular mechanism dissection suggested that AR in monocytes/macrophages upregulated the tumor necrosis factor-α, integrin β2, and lectin-type oxidized LDL receptor 1 molecules that are involved in 3 major inflammation-related processes in atherosclerosis, including monocytes/macrophages migration and adhesion to human umbilical vein ECs, and subsequent foam cell formation. Targeting AR via the AR degradation enhancer, ASC-J9, in wild-type-LDLR(-/-) mice showed similar effects as seen in monocyte/macrophage ARKO-LDLR(-/-) mice with little influence on lipid profile. In conclusion, the AR in monocytes/macrophages plays key roles in atherosclerosis and targeting AR with ASC-J9 may represent a new potential therapeutic approach to battle atherosclerosis.

The ABC of Thrombopoiesis

Platelets play an important role in atherogenesis and atherothrombosis. At early stages of atherosclerosis, platelet-borne chemokines and platelet–leukocyte complexes contribute to the recruitment of neutrophils and monocytes to inflamed arteries.1,2 At late stages, platelets orchestrate additional processes such as thrombosis by direct mechanisms and possibly by indirect mechanisms such as the induction of neutrophil extracellular traps.3,4 Although mechanisms of platelet-driven atherosclerosis have been studied widely, little is known about their production during atherosclerosis-promoting conditions. See accompanying article on page 751 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Murphy et al5 show that the lack of ABCB6 in the bone marrow leads to thrombocytosis, enhanced proinflammatory platelet activity, and accelerated atherosclerosis in atherosclerosis-prone hyperlipidemic low-density lipoprotein receptor–deficient mice (see the Figure). Interestingly, ABCB6 was found to be highly expressed in bone marrow …

The role of heat shock protein 90 in migration and proliferation of vascular smooth muscle cells in the development of atherosclerosis

The molecular chaperone heat shock protein 90 (HSP90) is overexpressed in plaques of atherosclerosis patients, and is associated with plaque instability. However, the role of HSP90 in atherosclerosis remains unclear. The present study investigated the effects of HSP90 inhibition on migration and proliferation of vascular smooth muscle cells (VSMCs) and involvement in atherosclerosis. To examine the role of HSP90 in VSMC migration, VSMCs were treated with the specific HSP90 inhibitors, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and STA-9090. Results of a chemotaxis assay showed that the HSP90 inhibitors suppress migration of VSMCs. HSP90 inhibition also prevented invasion and sprout formation of VSMCs via inhibition of matrix metalloproteinase-2 proteolytic activity. Results of a flow cytometric analysis showed that HSP90 inhibition induces cell cycle arrest via regulation of cyclin D3, PCNA and pRb. To investigate the role of HSP90 in the development of atherosclerosis, low-density lipoprotein receptor (LDLR) deficient mice were fed with a high cholesterol diet for 4weeks and treated with 17-AAG for 8weeks. HSP90 inhibition suppressed migration of VSMCs into atherosclerotic plaque lesions in high cholesterol diet-stimulated LDLR−/− mice. Inhibition of HSP90 attenuates formation of atherosclerotic plaques via suppression of VSMC migration and proliferation, indicating that HSP90 inhibitors can be used as therapeutic agents for atherosclerosis and in stent restenosis.

Intermittent hypoxia and hypercapnia induce pulmonary artery atherosclerosis and ventricular dysfunction in low density lipoprotein receptor deficient mice

Patients with obstructive sleep apnea, who experience episodic hypoxia and hypercapnia during sleep, often demonstrate increased inflammation, oxidative stress, and dyslipidemia. We hypothesized that sleep apnea patients would be predisposed to the development of atherosclerosis. To dissect the mechanisms involved, we developed an animal model in mice whereby we expose mice to intermittent hypoxia/hypercapnia (IHH) in normobaric environments. Two- to three-month-old low-density lipoprotein receptor deficient (Ldlr(-/-)) mice were fed a high-fat diet for 8 or 16 wk while being exposed to IHH for either 10 h/day or 24 h/day. Plasma lipid levels, pulmonary artery and aortic atherosclerotic lesions, and cardiac function were then assayed. Surprisingly, atherosclerosis in the aorta of IHH mice was similar compared with controls. However, in IHH mice, atherosclerosis was markedly increased in the trunk and proximal branches of the pulmonary artery of exposed mice; even though plasma cholesterol and triglycerides were lower than in controls. Hemodynamic analysis revealed that right ventricular maximum pressure and isovolumic relaxation constant were significantly increased in IHH exposed mice and left ventricular % fractional shortening was reduced. In conclusion, 1) Intermittent hypoxia/hypercapnia remarkably accelerated atherosclerotic lesions in the pulmonary artery of Ldlr(-/-) mice and 2) increased lesion formation in the pulmonary artery was associated with right and left ventricular dysfunction. These findings raise the possibility that patients with obstructive sleep apnea may be susceptible to atherosclerotic disease in the pulmonary vasculature, an observation that has not been previously recognized.

A Novel Mathematical Model Detecting Early Individual Changes of Insulin Resistance

Insulin resistance (IR) and hyperinsulinemia as well as obesity play a key role in the metabolic syndrome (MetS), type 2 diabetes (T2D), and associated cardiovascular disease. Unfortunately, IR and hyperinsulinemia are often diagnosed late (i.e., when the MetS is already clinically evident). An earlier diagnosis of IR would be desirable to reduce its clinical consequences, in particular in view of the increasing prevalence of obesity and diabetes conditions. For this purpose, we developed a mathematical model capable of detecting early onset of IR through small variations of insulin sensitivity, glucose effectiveness, and first- or second-phase responses. Murine models provide controlled conditions to study various stages of IR. Various degrees of hypercholesterolemia, obesity, IR, and atherosclerosis were induced in low-density lipoprotein receptor-deficient mice by feeding them cholesterol- or sucrose-rich diets. IR was assessed by oral glucose tolerance tests. Controls included animals fed exclusively, or switched back to, regular chow. A nonlinear mathematical model of the order of 5 was developed by refining Bergman's "Minimal Model" and then applied to experimental data. Different metabolic constellations consistently corresponded to specific and close-meshed changes in model parameters. Reduced second-phase glucose sensitivity characterized an early impaired glucose tolerance. Later stages showed an increased first-phase glucose sensitivity compensating for decreased insulin sensitivity. Finally, T2D was associated with both first- and second-phase sensitivities close to zero. The new mathematical model detected various insulin-sensitive or -resistant metabolic stages of IR. It can therefore be implemented for quantitative metabolic risk assessment and may be of therapeutic value by anticipating the start of therapeutic interventions.

GW24-e2397 Rosuvastatin could modulate insulin signalling and inhibit atherosclerosis beyond its plasma cholesterol-lowering effect in insulin-resistant mice

ObjectivesTo provide evidence that rosuvastatin could improve insulin-resistance and inhibit atherogenesis by modulating insulin signalling, and whether this effect beyond its plasma cholesterol-lowering effect.MethodsThirty-two 6-week-old low-density lipoprotein receptor deficient (LDLR-...

Pharmaceutical stabilization of mast cells attenuates experimental atherogenesis in low-density lipoprotein receptor-deficient mice

Mast cells (MCs) contribute to atherogenesis by releasing pro-inflammatory mediators to activate vascular cells and other inflammatory cells. This study examined whether MC activation or stabilization affects diet-induced atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr–/–) mice. When Ldlr–/– mice consumed an atherogenic diet for 3 or 6 months, MC activation with compound 48/80 (C48/80) increased aortic arch intima and total lesion areas, and plasma total cholesterol, LDL, and triglyceride levels, whereas MC stabilization with cromolyn reduced these parameters. There were significant differences in arch intima and total lesion areas, and plasma total cholesterol, LDL, and triglyceride levels between C48/80-treated and cromolyn-treated mice. To examine a therapeutic application of cromolyn in atherosclerosis, we fed Ldlr–/– mice an atherogenic diet for 3 months followed by giving mice cromolyn for additional 3 months. Cromolyn did not affect aortic arch intima area, but significantly reduced lipid deposition in the thoracic-abdominal aortas. In aortic arches, however, cromolyn treatment significantly reduced lesion contents of Mac-3+ macrophages, CD4+ T cells, activated MCs, and lesion cell proliferation. While plasma total cholesterol and LDL levels increased and high-density lipoprotein (HDL) levels decreased from 3 months to 6 months of an atherogenic diet, cromolyn treatment decreased significantly plasma total cholesterol, LDL, and triglyceride levels and increased HDL levels above those of 3-month time point. These observations demonstrate that MC stabilization reduces lesion inflammation, ameliorates plasma lipid profiles, and may serve as a potential therapy for this cardiovascular disease.

Atherosclerosis and cardiac function assessment in low-density lipoprotein receptor-deficient mice undergoing body weight cycling

Background:Obesity has become an epidemic in many countries and is supporting a billion dollar industry involved in promoting weight loss through diet, exercise and surgical procedures. Because of difficulties in maintaining body weight reduction, a pattern of weight cycling often occurs (so called ‘yo-yo' dieting) that may result in deleterious outcomes to health. There is controversy about cardiovascular benefits of yo-yo dieting, and an animal model is needed to better understand the contributions of major diet and body weight changes on heart and vascular functions. Our purpose is to determine the effects of weight cycling on cardiac function and atherosclerosis development in a mouse model.Methods:We used low-density lipoprotein receptor-deficient mice due to their sensitivity to metabolic syndrome and cardiovascular diseases when fed high-fat diets. Alternating ad libitum feeding of high-fat and low-fat (rodent chow) diets was used to instigate weight cycling during a 29-week period. Glucose tolerance and insulin sensitivity tests were done at 22 and 24 weeks, echocardiograms at 25 weeks and atherosclerosis and plasma lipoproteins assessed at 29 weeks.Results:Mice subjected to weight cycling showed improvements in glucose homeostasis during the weight loss cycle. Weight-cycled mice showed a reduction in the severity of atherosclerosis as compared with high-fat diet-fed mice. However, atherosclerosis still persisted in weight-cycled mice as compared with mice fed rodent chow. Cardiac function was impaired in weight-cycled mice and matched with that of mice fed only the high-fat diet.Conclusion:This model provides an initial structure in which to begin detailed studies of diet, calorie restriction and surgical modifications on energy balance and metabolic diseases. This model also shows differential effects of yo-yo dieting on metabolic syndrome and cardiovascular diseases.

KRP-203, Sphingosine 1-Phosphate Receptor Type 1 Agonist, Ameliorates Atherosclerosis in LDL-R −/− Mice

Sphingosine 1-phosphate (S1P) partly accounts for antiatherogenic properties of high-density lipoproteins. We previously demonstrated that FTY720, a synthetic S1P analog targeting all S1P receptors but S1P receptor type 2, inhibits murine atherosclerosis. Here, we addressed the identity of S1P receptor mediating atheroprotective effects of S1P. Low-density lipoprotein receptor-deficient mice on cholesterol-rich diet were given selective S1P receptor type 1 agonist KRP-203 (3.0 mg/kg per day; 6 and 16 weeks). KRP-203 substantially reduced atherosclerotic lesion formation without affecting plasma lipid concentrations. However, KRP-203 induced lymphopenia, reduced total (CD4(+), CD8(+)) and activated (CD69(+)/CD8(+), CD69(+)/CD4(+)) T cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased T-cell proliferation and interleukin-2 and interferon-γ production in activated splenocytes. Cyto- and chemokine (tumor necrosis factor-α, regulated and normal T cell expressed and secreted) levels in plasma and aortas were reduced by KRP-203 administration. Moreover, macrophages from KRP-203-treated mice showed reduced expression of activation marker MCH-II and poly(I:C)-elicited production of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6. In vitro studies demonstrated that KRP-203 reduced tumor necrosis factor-α, interleukin-6, and interferon-γ-induced protein-10 production; IκB and signal transducer and activator of transcription-1 phosphorylation; and nuclear factor κB and signal transducer and activator of transcription-1 activation in poly(I:C)-, lipopolysaccharide-, or interferon-γ-stimulated bone marrow macrophages, respectively. Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P receptor type 1 at least partially mediates antiatherogenic effects of S1P.

Abstract 130: Platelet Rap1 Signaling, Mediated by CalDAG-GEFI and P2Y12, Contributes to Atherosclerotic Lesion Development in Mice

Background and Objective The development and progression of atherosclerotic lesions is mediated by various blood cell types, including platelets and neutrophils. We recently identified CalDAG-GEFI (CDGI) as a critical component of integrin-mediated adhesion of platelets and neutrophils at sites of vascular injury. In platelets, signaling by CDGI mediates the early activation of Rap1, while receptor for ADP, P2Y12, the target of the clinically used drug Plavix, is required for sustained Rap1 activation and thrombus stability. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr-/-) mice lacking CDGI and/or P2Y12 in hematopoietic cells. Methods and Results Ldlr-/- mice were irradiated and reconstituted with bone-marrow from wild-type (WT), CalDAG-GEFI-/- (CdgI), P2y12-/-, or CdgI-/- P2y12-/- (DKO) mice. Reconstituted mice were fed a fat and cholesterol enriched diet (21% fat, 0.2% cholesterol) for 12 weeks, ad libitum. Atherosclerotic lesions in the aortic sinus of Ldlr-/-;CdgI-/- chimeras were 42% smaller than those in Ldlr-/-;WT controls (0.18 ± 0.02 mm2 vs 0.31 ± 0.05 mm2, respectively; n = 13 each group, p < 0.001). Lesions in Ldlr-/-;P2y12-/- chimeras were also significantly smaller compared to controls (0.22 ± 0.10 mm2; n = 13, p < 0.05). Lesions in Ldlr-/-;DKO were reduced by 48% compared to WT controls (0.16 ± 0.02 mm2; n = 13, p < 0.001 ), but they were not statistically different from Ldlr-/-;CdgI-/- chimeras. Platelet adhesion and activation on collagen under flow was markedly impaired in Ldlr-/-;CdgI-/- and Ldlr-/-;DKO blood, but only partially impaired in Ldlr-/-;P2y12-/- blood. Importantly, firm neutrophil adhesion to collagen-bound platelets was significantly reduced in Ldlr-/-;CdgI-/- and Ldlr-/-;DKO blood, but not in Ldlr-/-;P2y12-/- blood. Total cholesterol and triglyceride levels were similar among groups. Conclusion Our findings reveal a critical role for CDGI and P2Y12, and Rap1-dependent platelet activation, in promoting atherosclerotic lesion development in hypercholesterolemic mice. Further studies are required to determine if the observed protection in Ldlr-/-;CdgI-/- chimeras is due to impaired neutrophil function.

Abstract 243: Adiponectin Inhibits Angiotensin II-induced Abdominal Aortic Aneurysm Formation

BACKGROUND Abdominal aortic aneurysms (AAA) are characterized by inflammation, loss of smooth muscle cells (SMC) and degradation of extracellular matrix in the vessel wall. Adiponectin, an adipokine produced by adipose tissue, exerts profound anti-inflammatory and anti-atherogenic effects on vascular cells. However, the role of adiponectin on AAA has not been studied. We investigated the impact of increased plasma adiponectin levels on angiotensin II (AngII)-induced AAA formation in mice. METHOD AND RESULTS Male low-density lipoprotein receptor-deficient (LDLR -/- ) mice fed on a high-fat diet were infused with AngII (25 mg/kg/min) for 8 weeks to induce AAA. Mice infused with saline was used as controls. A recombinant adenoviral vector encoding mouse adiponectin (Ad-APN) was injected intravenously to induce adiponectin expression. A recombinant adenovirus expressing green fluorescence protein (GFP) was used as a control (Ad-GFP). Eight weeks after injection, plasma adiponectin levels were 10-fold higher (253.9±59.0 μg/mL) in Ad-APN mice compared to Ad-GFP mice (25.2±3.8 μg/mL, P<0.001). Aortic diameter was significantly reduced in Ad-APN mice compared to Ad-GFP mice (60% reduction). This was accompanied by decreased frequency and size of abdominal atherosclerotic lesions in Ad-APN mice compared to Ad-GFP mice. Histologically, elastin was degraded in all AngII-infused mice compared to saline-infused mice. However elastin was more preserved in Ad-APN mice compared to Ad-GFP mice (P<0.05). Gene expression analysis of the abdominal aorta revealed a decrease of CD3e (P<0.05) but no profound changes in inflammatory genes, IL-6, TNF-α and CD68. Decreased mRNA expression of the SMC marker SM22α was observed in all AngII-infused mice compared to saline-infused mice. Expression of SM22α was two-fold higher in the abdominal aorta in Ad-APN mice than in Ad-GFP mice (P<0.05). CONCLUSION These results provide first evidence that adiponectin overexpression significantly inhibits AngII-induced AAA formation in LDLR -/- mice. The protective actions of adiponectin appear to be mediated, in part, through preservation of SMC content and elastin in the vascular wall. The mechanisms of adiponectin inhibiting AAA formation deserve further attention.

Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular inflammation

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.

Effects of lifestyle modifications on cognitive impairments in a mouse model of hypercholesterolemia

Epidemiological studies indicate that high midlife plasma cholesterol levels increases the risk of Alzheimer's disease. Moreover, middle-aged familial hypercholesterolemia (FH) subjects show a particularly high incidence of mild cognitive impairments (MCI). These evidence points to hypercholesterolemia as one of the modifiable risk factors focused on prevention/treatment of cognitive deterioration. The present study draws a comparison between pharmacological (lipid-lowering drug probucol) and non-pharmacological (voluntary running wheel, RW) approaches for the management of hypercholesterolemia and cognitive impairments associated with the low-density lipoprotein receptor-deficient (LDLr−/−) mice, a well-established rodent model of FH. We also investigated whether exposure to environmental enrichment (EE), a feasible option to increase physical activity in young mice cohort, from birth to adolescence (PN45) yields long-term behavioral changes in adult LDLr−/− mice (PN90). We observed that both probucol and RW significantly decreased total and non-HDL plasma cholesterol levels in LDLr−/− mice. Notably, only physical exercise mitigated the spatial memory deficits of LDLr−/− mice. In addition, we showed that exposure to EE from birth until the adolescence did not mitigate the spatial memory deficits of adult LDLr−/− mice in the object location task, although it induced persistent anxyolitic-like effects in the open field arena. Collectively, our results emphasize the advantages physical exercise, in comparison to lipid-lowering drugs, for the management of cognitive deficits associated with FH.

Enhanced adiponectin actions by overexpression of adiponectin receptor 1 in macrophages

ObjectiveAdiponectin is one of several important, metabolically active cytokines secreted from adipose tissue. Epidemiologic studies have associated low circulating levels of this adipokine with multiple metabolic disorders including obesity, insulin resistance, type II diabetes, and cardiovascular disease. To investigate how enhanced adiponectin-mediated changes in metabolism in vivo, we generated transgenic mice which specifically overexpress the gene coding for adiponectin receptor 1 (AdipoR1) in mouse macrophages using the human scavenger receptor A-I gene (SR-AI) enhancer/promoter. We found that macrophage-specific AdipoR1 transgenic mice (AdR1-TG) presented reduced whole body weight, fat accumulation and liver steatosis when these transgenic mice were fed with a high fat diet. Moreover, these macrophage AdR1-TG mice exhibited enhanced whole-body glucose tolerance and insulin sensitivity with reduced proinflammatory cytokines, MCP-1 and TNF-α, both in the serum and in the insulin target metabolic tissues. Additional studies demonstrated that these macrophage AdR1-TG animals exhibited reduced macrophage foam cell formation in the arterial wall when these transgenic mice were crossed with a low-density lipoprotein receptor (Ldlr) deficient mouse model. ConclusionsThese results suggest that AdipoR1 overexpressed in macrophages can physiologically modulate metabolic activities in vivo by enhancing adiponectin actions in distal metabolically active tissues. The AdipoR1 modified macrophages provide unique interactions with the residented tissues/cells, suggesting a novel role of macrophage adiponectin receptor in improving metabolic disorders in vivo.

Carbamyl Adducts on Low-Density Lipoprotein Induce IgG Response inLDLR−/−Mice and Bind Plasma Autoantibodies in Humans Under Enhanced Carbamylation

Post-translational modification of proteins via carbamylation predicts increased risk for coronary artery disease. Uremia and smoke exposure are known to increase carbamylation. The aim was to investigate the role of carbamylated low-density lipoprotein (LDL) immunization on antibody formation and atherogenesis in LDL receptor-deficient (LDLR-/-) mice, and to study autoantibodies to carbamylated proteins in humans with carbamylative load. LDLR-/- mice immunized with carbamylated mouse LDL (msLDL; n=10) without adjuvant showed specific immunoglobulin G (IgG) antibody levels to carbamyl-LDL and malondialdehyde-modified LDL (MDA-LDL) but not to oxidized LDL or native LDL. Immunization did not influence the atherosclerotic plaque area compared with control LDLR-/- mice immunized with native msLDL (n=10) or phosphate-buffered saline (n=11). Humans with high plasma urea levels, as well as smokers, had increased IgG autoantibody levels to carbamyl-modified proteins compared to the subjects with normal plasma urea levels, or to nonsmokers. Carbamyl-LDL induced specific IgG antibody response cross-reactive with MDA-LDL in mice. IgG antibodies to carbamyl-LDL were also found in human plasma and related to conditions known to have increased carbamylation, such as uremia and smoking. Plasma antibodies to carbamylated proteins may serve as new indicator of in vivo carbamylation. These data give insight into mechanisms of in vivo humoral recognition of post-translationally modified structures. Humoral IgG immune response to carbamylated proteins is suggested to play a role in conditions leading to enhanced carbamylation, such as uremia and smoking.

Detection of early stage atherosclerotic plaques using PET and CT fusion imaging targeting P-selectin in low density lipoprotein receptor-deficient mice

BackgroundSensitive detection and qualitative analysis of atherosclerotic plaques are in high demand in cardiovascular clinical settings. The leukocyte–endothelial interaction mediated by an adhesion molecule P-selectin participates in arterial wall inflammation and atherosclerosis. Methods and resultsA 64Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated anti-P-selectin monoclonal antibody (64Cu-DOTA-anti-P-selectin mAb) probe was prepared by conjugating an anti-P-selectin monoclonal antibody with DOTA followed by 64Cu labeling. Thirty-six hours prior to PET and CT fusion imaging, 3MBq of 64Cu-DOTA-anti-P-selectin mAb was intravenously injected into low density lipoprotein receptor-deficient Ldlr-/- mice. After a 180min PET scan, autoradiography and biodistribution of 64Cu-DOTA-anti-P-selectin monoclonal antibody was examined using excised aortas. In Ldlr-/- mice fed with a high cholesterol diet for promotion of atherosclerotic plaque development, PET and CT fusion imaging revealed selective and prominent accumulation of the probe in the aortic root. Autoradiography of aortas that demonstrated probe uptake into atherosclerotic plaques was confirmed by Oil red O staining for lipid droplets. In Ldlr-/- mice fed with a chow diet to develop mild atherosclerotic plaques, probe accumulation was barely detectable in the aortic root on PET and CT fusion imaging. Probe biodistribution in aortas was 6.6-fold higher in Ldlr-/- mice fed with a high cholesterol diet than in those fed with a normal chow diet. 64Cu-DOTA-anti-P-selectin mAb accumulated selectively in aortic atherosclerotic plaques and was detectable by PET and CT fusion imaging in Ldlr-/- mice. ConclusionsP-selectin is a candidate target molecule for early-phase detection by PET and CT fusion imaging of atherosclerotic plaques.

Toll-like receptor 3 and 4 signalling through the TRIF and TRAM adaptors in haematopoietic cells promotes atherosclerosis

Members of the Toll-like receptor (TLR) family initiate innate immune responses and were recently shown to play a role in atherosclerosis. However, the mechanisms that link TLR ligation to vascular inflammation and atherogenesis remain unclear. To identify which signalling pathways downstream of TLRs in immune cells are pro-atherogenic, we analysed the role of the TLR-specific adaptors MyD88 adaptor-like (MAL), TRIF-related adaptor molecule (TRAM), and TIR-domain-containing adaptor-inducing interferon-β (TRIF) in atherosclerosis. Using a bone-marrow transplantation strategy into low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, we could specifically study the absence of the TLR adaptors in immune cells. We showed that haematopoietic deficiency of TRAM and TRIF, but not MAL, reduces atherosclerosis without affecting cholesterol metabolism. This was mediated by decreased aortic inflammation, indicated by lower aortic levels of pro-inflammatory mediators, and reduced influx of macrophages and T cells. Furthermore, by studying Tlr3(-/-) chimeric Ldlr(-/-) mice, we found that deleting TLR3 in immune cells significantly reduced both aortic inflammation and atherosclerotic burden. By studying hypercholesterolaemic mice with defects in TLR-signalling adaptors, we demonstrated that deleting either TRAM or TRIF in immune cells is sufficient to attenuate vessel inflammation and protect against atherosclerosis. In addition, these adaptors elicit partly different sets of inflammatory mediators and can independently inhibit the disease process. Furthermore, we identify TLR3 as a pro-atherogenic receptor in haematopoietic immune cells. The identification of these pro-atherogenic pathways downstream of TLR3 and TLR4 contributes to a better understanding of TLRs and their signalling pathways in the pathogenesis of atherosclerosis.

Targeting GGTase-I Activates RHOA, Increases Macrophage Reverse Cholesterol Transport, and Reduces Atherosclerosis in Mice

Statins have antiinflammatory and antiatherogenic effects that have been attributed to inhibition of RHO protein geranylgeranylation in inflammatory cells. The activity of protein geranylgeranyltransferase type I (GGTase-I) is widely believed to promote membrane association and activation of RHO family proteins. However, we recently showed that knockout of GGTase-I in macrophages activates RHO proteins and proinflammatory signaling pathways, leading to increased cytokine production and rheumatoid arthritis. In this study, we asked whether the increased inflammatory signaling of GGTase-I-deficient macrophages would influence the development of atherosclerosis in low-density lipoprotein receptor-deficient mice. Aortic lesions in mice lacking GGTase-I in macrophages (Pggt1b▵/▵) contained significantly more T lymphocytes than the lesions in controls. Surprisingly, however, mean atherosclerotic lesion area in Pggt1b▵/▵ mice was reduced by ≈60%. GGTase-I deficiency reduced the accumulation of cholesterol esters and phospholipids in macrophages incubated with minimally modified and acetylated low-density lipoprotein. Analyses of GGTase-I-deficient macrophages revealed upregulation of the cyclooxygenase 2-peroxisome proliferator-activated-γ pathway and increased scavenger receptor class B type I- and CD36-mediated basal and high-density lipoprotein-stimulated cholesterol efflux. Lentivirus-mediated knockdown of RHOA, but not RAC1 or CDC42, normalized cholesterol efflux. The increased cholesterol efflux in cultured cells was accompanied by high levels of macrophage reverse cholesterol transport and slightly reduced plasma lipid levels in vivo. Targeting GGTase-I activates RHOA and leads to increased macrophage reverse cholesterol transport and reduced atherosclerosis development despite a significant increase in inflammation.