LDL Receptor-deficient Mice Research Articles

OP0129 Ldl Cholesterol-Accumulation during Experimental OA Leads to Increased Synovial Thickening and Ectopic Bone Formation, While Excessive Cholesterol Levels Shift the Balance towards Cartilage Destruction

BackgroundA relation between osteoarthritis (OA) and the metabolic syndrome has long been established. One of the characteristics of the metabolic syndrome is increased cholesterol levels. In a recent study, we...

223 A Novel Immunoglobulin G Autoantibody Against Low Density Lipoprotein (LDL) with Pathogenic Functions

IntroductionIgG autoantibodies reacting with different forms of modified LDL are found in blood and have been associated with cardiovascular events related to atherosclerosis. However there is a controversy as to...

Abstract 474: Biglycan Deficiency is Not Protective Against the Development of Atherosclerosis

Atherosclerosis is the leading cause of death worldwide. As outlined in the Response to Retention hypothesis proteoglycan mediated lipid retention in the vascular wall is thought to be a major cause of atherosclerosis. We and others demonstrated co-localization of the vascular wall matrix proteoglycan biglycan with apoB containing lipoproteins within lesions. Furthermore, we have previously shown that increased biglycan leads to increased atherosclerosis. Thus we hypothesized that biglycan deficiency would attenuate the development of atherosclerosis. Biglycan deficient mice on a mixed genetic background were crossed with LDL receptor deficient mice (C57Bl6). Biglycan deficient and wildtype LDLr-/- mice were fed a 0.12% cholesterol diet for 26 weeks. There were no differences in blood glucose, blood pressure, or triglycerides between genotypes. Total cholesterol progressively increased in both groups over the course of the study and was significantly higher in biglycan deficient compared to wildtype mice at 26 weeks (p<0.05). Biglycan has been proposed to play a role in regulation of TGF-β levels and activity. We previously demonstrated that aged biglycan deficient mice had increased TGF-β levels compared to wildtype. TGF-β was significantly increased in biglycan deficient mice prior to study while fed chow and steadily increased with the high cholesterol diet over the course of the study in both genotypes (p<0.001); however, biglycan deficient mice had significantly more TGF-β at each timepoint compared to wildtype controls (p<0.001). Surprisingly, analysis of the aortic intimal surface revealed a 1.4 fold increase in atherosclerosis in biglycan deficient mice compared to wildtype controls (p=0.07). Thus in opposition to our hypothesis, biglycan deficiency is not protective against the development of atherosclerosis and may exacerbate disease development. Elevated TGF-β and/or cholesterol in biglycan deficient mice may contribute to the increased atherosclerosis observed but further research is required to elicit the mechanism.

Abstract 366: Inhibition of Interleukin-1ß by a Monoclonal Antibody Therapy Reduces Vascular Calcification in Ldlr-/- Mice

Objective: Given the link between cholesterol and activation of inflammation via interleukin-1β, we thus tested the effects of IL-1β inhibition on atherosclerotic calcification in mice. Methods and Results: A mouse monoclonal antibody (mAB) against IL-1β or placebo was administered subcutaneously to Ldlr-/- and Tg(Pcsk9) models fed a Western diet. Drug level, anthropometric, lipid and glucose profiles were determined. PCSK9, SAA1 and cytokine expressions were measured by ELISA. Aortic calcification was determined by micro-CT and X-Ray densitometry and aortic flow velocity was assessed by ultrasound. Circulating levels of IL-1β in Ldlr-/- mice were significant twice that observed in Tg(Pcsk9) mice. Both mAb and placebo treated mice did not differ in their growth, lipid, glucose profiles and other cytokines while plasma SAA1 levels were lower in mAb-treated mice. Calcifications were significantly diminished in mAb-treatment Ldlr-/- mice (a reduction of 75% by X-ray and 96% by micro-CT) and reduced insignificantly in mAb-treatment Tg(Pcsk9) mice, whereas aortic flow velocity was unchanged in both models. Conclusions: Herein we demonstrate that aortic calcifications can be inhibited by IL-1β mAb in LDL-receptor deficient mice. These results have a translational component to prevent vascular calcification in human and represent new evidence to rationalize targeting inflammation in cardiovascular disease.

Abstract 11: Macrophage Expression of the Notch Ligand Delta-Like 4 Promotes Vein Graft Disease in LDL Receptor--Deficient Mice

Background: Vein graft failure causes devastating complications in patients with peripheral arterial disease or ischemic heart disease, but its underlying mechanisms remain obscure and no effective therapeutic measures are available. Methods and Results: We tested the hypothesis that Notch signaling triggered by its ligand Delta-like 4 (Dll4) promotes macrophage activation and vein graft disease. Vein graft surgery was performed in high-fat fed LDL receptor-deficient (Ldlr-/-) mice by implanting donor inferior vena cava into recipient right carotid arteries. [Approach 1: Dll4 antibody] Dll4 blocking antibody was administered for 28 days. Dll4 blockade inhibited lesion development and macrophage accumulation (Figures) in vein grafts, and suppressed macrophage expression of genes typical of pro-inflammatory M1 macrophages (e.g., IL-1β, TNF-α). In vivo molecular imaging demonstrated that Dll4 antibody treatment suppressed MMP activity in lesional macrophages. Dll4 blockade concomitantly attenuated collagen thinning. [Approach 2: siRNA delivery to macrophages] To address the relative contribution of macrophages to Dll4-mediated vein graft disease in vivo, we delivered Dll4 siRNA oligos encapsulated in macrophage-targeted lipid nanoparticles. In vivo Dll4 silencing in macrophages reduced lesion development and macrophage burden in vein grafts of Ldlr-/- mice to a similar extent as those of Dll4 antibody therapy. In vitro gain-of-function and loss-of-function studies suggested that Dll4 promotes expression of pro-inflammatory molecules in macrophages. Furthermore, macrophage Dll4 stimulated smooth muscle cell (SMC) proliferation and migration and suppressed their differentiation. Conclusions: These results suggest the novel mechanism that macrophage Dll4 promotes vein graft lesion development by exacerbating inflammation and crosstalk between macrophages and SMC, supporting the Dll4-Notch axis as a potential therapeutic target.

Increased atherosclerosis in mice with increased vascular biglycan content

ObjectiveThe response to retention hypothesis of atherogenesis proposes that atherosclerosis is initiated via the retention of atherogenic lipoproteins by vascular proteoglycans. Co-localization studies suggest that of all the vascular proteoglycans, biglycan is the one most closely co-localized with LDL. The goal of this study was to determine if over-expression of biglycan in hyperlipidemic mice would increase atherosclerosis development. MethodsTransgenic mice were developed by expressing biglycan under control of the smooth muscle actin promoter, and were crossed to the LDL receptor deficient (C57BL/6 background) atherosclerotic mouse model. Biglycan transgenic and non-transgenic control mice were fed an atherogenic Western diet for 4–12 weeks. ResultsLDL receptor deficient mice overexpressing biglycan under control of the smooth muscle alpha actin promoter had increased atherosclerosis development that correlated with vascular biglycan content. ConclusionIncreased vascular biglycan content predisposes to increased lipid retention and increased atherosclerosis development.

Group 1B phospholipase A2 inactivation suppresses atherosclerosis and metabolic diseases in LDL receptor-deficient mice

ObjectivePrevious studies have shown that inactivation of the group 1B phospholipase A2 (Pla2g1b) suppresses diet-induced obesity, hyperglycemia, insulin resistance, and hyperlipidemia in C57BL/6 mice. A possible influence of Pla2g1b inactivation on atherosclerosis has not been addressed previously. The current study utilized LDL receptor-deficient (Ldlr−/−) mice with plasma lipid levels and distribution similar to hyperlipidemic human subjects as a preclinical animal model to test the effectiveness of Pla2g1b inactivation on atherosclerosis. Methods and resultsThe Pla2g1b+/+Ldlr−/− and Pla2g1b−/−Ldlr−/− mice were fed a low fat chow diet or a hypercaloric diet with 58.5 kcal% fat and 25 kcal% sucrose for 10 weeks. Minimal differences were observed between Pla2g1b+/+Ldlr−/− and Pla2g1b−/−Ldlr−/− mice when the animals were maintained on the low fat chow diet. However, when the animals were maintained on the hypercaloric diet, the Pla2g1+/+Ldlr−/− mice showed the expected body weight gain but the Pla2g1b−/−Ldlr−/− mice were resistant to diet-induced body weight gain. The Pla2g1b−/−Ldlr−/− mice also displayed lower fasting glucose, insulin, and plasma lipid levels compared to the Pla2g1b+/+Ldlr−/− mice, which displayed robust hyperglycemia, hyperinsulinemia, and hyperlipidemia in response to the hypercaloric diet. Importantly, atherosclerotic lesions in the aortic roots were also reduced 7-fold in the Pla2g1b−/−Ldlr−/− mice. ConclusionThe effectiveness of Pla2g1b inactivation to suppress diet-induced body weight gain and reduce diabetes and atherosclerosis in LDL receptor-deficient mice suggests that pharmacological inhibition of Pla2g1b may be a viable strategy to decrease diet-induced obesity and the risk of diabetes and atherosclerosis in humans.

Testing the Role of Myeloid Cell Glucose Flux in Inflammation and Atherosclerosis

Inflammatory activation of myeloid cells is accompanied by increased glycolysis, which is required for the surge in cytokine production. Although in vitro studies suggest that increased macrophage glucose metabolism is sufficient for cytokine induction, the proinflammatory effects of increased myeloid cell glucose flux in vivo and the impact on atherosclerosis, a major complication of diabetes, are unknown. We therefore tested the hypothesis that increased glucose uptake in myeloid cells stimulates cytokine production and atherosclerosis. Overexpression of the glucose transporter GLUT1 in myeloid cells caused increased glycolysis and flux through the pentose phosphate pathway but did not induce cytokines. Moreover, myeloid-cell-specific overexpression of GLUT1 in LDL receptor-deficient mice was ineffective in promoting atherosclerosis. Thus, increased glucose flux is insufficient for inflammatory myeloid cell activation and atherogenesis. If glucose promotes atherosclerosis by increasing cellular glucose flux, myeloid cells do not appear to be the key targets.

Hyperhomocysteinemia independently causes and promotes atherosclerosis in LDL receptor-deficient mice.

BackgroundHyperhomocysteine is an independent risk factor of coronary heart disease (CHD). However, whether hyperhomocysteine affects the progression of atherosclerosis is unclear. In the present study, we examined the effect of hyperhomocysteine on the formation of atherosclerosis in low-density lipoprotein receptor-deficient (LDLr−/−) mice.MethodsForty-eight 7-week-old LDLr−/− mice were assigned to the following groups: mice fed a standard rodent diet (control group), mice fed a high-methionine diet (high-methionine group), mice fed a high-fat diet (high-fat group), and mice fed a diet high in both methionine and fat (high-methionine and high-fat group). At the age of 19, 23, and 27 weeks, four mice at each interval in every group were sacrificed.ResultsAt the end of the study, mice did not show atherosclerotic lesions in the aortic sinus and aortic surface until 27 weeks old in the control group. However, atherosclerotic lesions developed in the other three groups at 19 weeks. The amount of atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group (P < 0.001). Atherosclerotic lesions on the aortic surface in the high-methionine and high-fat group were the most severe. The mean area of atherosclerotic lesions in the aortic sinus compared with atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group (P < 0.001). Atherosclerotic lesions in the aortic sinus in the high-methionine and high-fat group were the most severe.ConclusionsHomocysteinemia accelerates atherosclerotic lesions and induces early atherosclerosis independently in LDLr−/− mice. Reducing the level of homocysteinemia may be beneficial for prevention and treatment of CHD.

A9.13 systemic LDL cholesterol-accumulation during experimental oa leads to increased synovial thickening, s100a8/9 production and ectopic bone formation

IntroductionIn a previous study, we showed that LDL accumulation by LDL receptor deficient mice resulted in increased ectopic bone formation during experimental osteoarthritis (OA). Furthermore, we found that S100A8/A9 proteins...

MafB promotes atherosclerosis by inhibiting foam-cell apoptosis

MafB is a transcription factor that induces myelomonocytic differentiation. However, the precise role of MafB in the pathogenic function of macrophages has never been clarified. Here we demonstrate that MafB promotes hyperlipidemic atherosclerosis by suppressing foam-cell apoptosis. Our data show that MafB is predominantly expressed in foam cells found within atherosclerotic lesions, where MafB mediates the oxidized LDL-activated LXR/RXR-induced expression of apoptosis inhibitor of macrophages (AIM). In the absence of MafB, activated LXR/RXR fails to induce the expression of AIM, a protein that is normally responsible for protecting macrophages from apoptosis; thus, Mafb-deficient macrophages are prone to apoptosis. Haematopoietic reconstitution with Mafb-deficient fetal liver cells in recipient LDL receptor-deficient hyperlipidemic mice revealed accelerated foam-cell apoptosis, which subsequently led to the attenuation of the early atherogenic lesion. These findings represent the first evidence that the macrophage-affiliated MafB transcription factor participates in the acceleration of atherogenesis.

An accelerated mouse model for atherosclerosis and adipose tissue inflammation

BackgroundObesity and particularly the metabolic syndrome, which is often associated with obesity, combine a major risk for type 2 diabetes and cardiovascular disease. Emerging evidence indicate obesity-associated subclinical inflammation primarily originating from adipose tissue as a common cause for type 2 diabetes and cardiovascular disease. However, a suitable and well-characterized mouse model to simultaneously study obesity-associated metabolic disorders and atherosclerosis is not available yet. Here we established and characterized a murine model combining diet-induced obesity and associated adipose tissue inflammation and metabolic deteriorations as well as atherosclerosis, hence reflecting the human situation of cardio-metabolic disease.MethodsWe compared a common high-fat diet with 0.15% cholesterol (HFC), and a high-fat, high-sucrose diet with 0.15% cholesterol (HFSC) fed to LDL receptor-deficient (LDLR-/-) mice. Insulin resistance, glucose tolerance, atherosclerotic lesion formation, hepatic lipid accumulation, and inflammatory gene expression in adipose tissue and liver were assessed.ResultsAfter 12–16 weeks, LDLR-/- mice fed HFSC or HFC developed significant diet-induced obesity, adipose tissue inflammation, insulin resistance, and impaired glucose tolerance compared to lean controls. Notably, HFSC-fed mice developed significantly higher adipose tissue inflammation in parallel with significantly elevated atherosclerotic lesion area compared to those on HFC. Moreover, LDLR-/- mice on HFSC showed increased insulin resistance and impaired glucose tolerance relative to those on HFC. After prolonged feeding (20 weeks), however, no significant differences in inflammatory and metabolic parameters as well as atherosclerotic lesion formation were detectable any more between LDLR-/- mice fed HFSC or HFC.ConclusionThe use of high sucrose rather than more complex carbohydrates in high-fat diets significantly accelerates development of obesity-driven metabolic complications and atherosclerotic plaque formation parallel to obesity-induced adipose tissue inflammation in LDLR-/- mice. Hence LDLR-/- mice fed high-fat high-sucrose cholesterol-enriched diet appear to be a suitable and time-saving animal model for cardio-metabolic disease. Moreover our results support the suggested interrelation between adipose tissue inflammation and atherosclerotic plaque formation.

Regression of atherosclerosis with anti-CD3 antibody via augmenting a regulatory T-cell response in mice

Although recent animal studies have investigated the cellular and molecular mechanisms underlying the process of atherosclerosis regression, it remains unknown whether adaptive immune responses including T cells are involved in this process. We investigated the role of T cells in atherosclerosis regression. LDL receptor-deficient mice were fed a high-cholesterol diet for 8 weeks to form atherosclerotic lesions and were then changed to a standard diet, and atherosclerosis was assessed 4 weeks later. Just before changing the diet, the mice received an iv injection of anti-CD3 antibody (CD3-Ab) or control immunoglobulin G for 5 consecutive days. CD3-Ab treatment regressed atherosclerosis and decreased the accumulation of macrophages and CD4(+) T cells in the plaques. CD3-Ab treatment also dramatically reduced CD4(+) T cells and increased the proportion of regulatory T cells (Tregs). Depletion of Tregs by anti-CD25 antibody injection abolished the regression of atherosclerosis seen in CD3-Ab-treated mice, indicating the essential role for Tregs in this process. CD3-Ab treatment induced rapid regression of established atherosclerosis via reducing CD4(+) T cells and increasing the proportion of Tregs. These findings suggest that therapeutic intervention for T-cell-mediated immune responses may represent a novel strategy to induce atherosclerosis regression in combination with lipid-lowering therapy.

CER-001, a HDL-mimetic, stimulates the reverse lipid transport and atherosclerosis regression in high cholesterol diet-fed LDL-receptor deficient mice

ObjectiveCER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and phospholipids that was designed to mimic the beneficial properties of nascent pre-β HDL. In this study, we have evaluated the capacity of CER-001 to perform reverse lipid transport in single dose studies as well as to regress atherosclerosis in LDLr−/− mice after short-term multiple-dose infusions. Approach and resultsCER-001 induced cholesterol efflux from macrophages and exhibited anti-inflammatory response similar to natural HDL. Studies with HUVEC demonstrated CER-001 at a concentration of 500μg/mL completely suppressed the secretion of cytokines IL-6, IL-8, GM-CSF and MCP-1. Following infusion of CER-001 (10mg/kg) in C57Bl/6J mice, we observed a transient increase in the mobilization of unesterified cholesterol in HDL particles containing recombinant human apoA-I. Finally we show that cholesterol elimination was stimulated in CER-001 treated animals as demonstrated by the increased cholesterol concentration in liver and feces. In a familial hypercholesterolemia mouse model (LDL-receptor deficient mice), the infusion of CER-001 caused 17% and 32% reductions in plaque size, 17% and 23% reductions in lipid content after 5 and 10 doses given every 2 days, respectively. Also, there was an 80% reduction in macrophage content in the plaque following 5 doses, and decreased VCAM-1 expression by 16% and 22% in the plaque following 5 and 10 intravenous doses of CER-001, respectively. ConclusionThese data demonstrate that CER-001 rapidly enhances reverse lipid transport in the mouse, reducing vascular inflammation and promoting regression of diet-induced atherosclerosis in LDLr−/− mice upon a short-term multiple dose treatment.

Curcumin modulation of high fat diet-induced atherosclerosis and steatohepatosis in LDL receptor deficient mice

ObjectiveConsuming curcumin may benefit health by modulating lipid metabolism and suppressing atherogenesis. Fatty acid binding proteins (FABP-4/aP2) and CD36 expression are key factors in lipid accumulation in macrophages and foam cell formation in atherogenesis. Our earlier observations suggest that curcumin's suppression of atherogenesis might be mediated through changes in aP2 and CD36 expression in macrophages. Thus, this study aimed to further elucidate the impact of increasing doses of curcumin on modulation of these molecular mediators on high fat diet-induced atherogenesis, inflammation, and steatohepatosis in Ldlr−/− mice. MethodsLdlr−/− mice were fed low fat (LF) or high fat (HF) diet supplemented with curcumin (500 HF + LC; 1000 HF + MC; 1500 HF + HC mg/kg diet) for 16 wks. Fecal samples were analyzed for total lipid content. Lipids accumulation in THP-1 cells and expression of aP2, CD36 and lipid accumulation in peritoneal macrophages were measured. Fatty streak lesions and expression of IL-6 and MCP-1 in descending aortas were quantified. Aortic root was stained for fatty and fibrotic deposits and for the expression of aP2 and VCAM-1. Total free fatty acids, insulin, glucose, triglycerides, and cholesterol as well as several inflammatory cytokines were measured in plasma. The liver's total lipids, cholesterol, triglycerides, and HDL content were measured, and the presence of fat droplets, peri-portal fibrosis and glycogen was examined histologically. ResultsCurcumin dose-dependently reduced uptake of oxLDL in THP-1 cells. Curcumin also reduced body weight gain and body fat without affecting fat distribution. During early intervention, curcumin decreased fecal fat, but at later stages, it increased fat excretion. Curcumin at medium doses of 500–1000 mg/kg diet was effective at reducing fatty streak formation and suppressing aortic expression of IL-6 in the descending aorta and blood levels of several inflammatory cytokines, but at a higher dose (HF + HC, 1500 mg/kg diet), it had adverse effects on some of these parameters. This U-shape like trend was also present when aortic root sections were examined histologically. However, at a high dose, curcumin suppressed development of steatohepatosis, reduced fibrotic tissue, and preserved glycogen levels in liver. ConclusionCurcumin through a series of complex mechanisms, alleviated the adverse effects of high fat diet on weight gain, fatty liver development, dyslipidemia, expression of inflammatory cytokines and atherosclerosis in Ldlr−/− mouse model of human atherosclerosis. One of the mechanisms by which low dose curcumin modulates atherogenesis is through suppression of aP2 and CD36 expression in macrophages, which are the key players in atherogenesis. Overall, these effects of curcumin are dose-dependent; specifically, a medium dose of curcumin in HF diet appears to be more effective than a higher dose of curcumin.

LMP7 immunoproteasome subunit deficiency does not alter early atherosclerosis in LDL receptor deficient mice

Purpose: Accumulating evidence suggests that the ubiquitin-proteasome system (UPS) plays an important role in atherogenesis. The immunoproteasome is the predominant proteasome in monocytes and macrophages. Its proteolytic subunit LMP7 is induced by inflammatory cytokines. Besides its role in antigen presentation the immunoproteasome is involved in protein homeostasis under conditions of oxidative stress, regulation of cytokine expression and cell proliferation. The current study evaluates the effect of LMP7 deficiency on early atherosclerosis in LDL receptor deficient (LDLR -/-) mice. Methods and results: For investigation of early atherosclerosis LDLR and LMP7 double knock-out (LDLR-/- LMP7-/-) and LDLR-/- mice, 10 weeks of age, were fed a Western type diet for 6 weeks. There were no significant differences in body weight or general condition of the animals. Cholesterol and triglyceride plasma levels were similar in both groups. LDLR-/- LMP7-/- mice exhibited significantly lower systolic blood pressure compared to LDLR-/- mice (113.8±2.8 vs. 128.1±1.6 mmHg; p < 0.05). Importantly, there were no differences in early atherosclerotic lesion burden as assessed by en face Oil red O staining of whole aortas and aortic root sections. Serum cytokine levels of IL-6, MCP-1 and IFN-γ did not differ significantly between both groups. Functional integrity of the UPS was demonstrated by the absence of accumulation of polyubiquitinated proteins in LDLR-/- LMP7-/- compared to LDLR-/- mice. Unchanged rates of protein oxidation in LDLR-/- LMP7-/- as assessed by OxyBlot demonstrated sufficient elimination of damaged proteins and/or preserved anti-oxidative capacity in aortas. Conclusion: LMP7 immunoproteasome subunit deficiency does not alter early atherosclerotic lesion progression, demonstrating functional integrity of a LMP7-deficient UPS in early atherogenesis. Future studies will investigate the effect of LMP7 deficiency in advanced stages of atherosclerosis.

The Near Infra-Red (NIRF) molecular imaging of oxidised LDL in atherosclerosis with the native autoantibody LO1, and its molecularly expressed cysteine-tagged Fab construct (LO1-Fab-cys)

Purpose: Oxidation of low density lipoprotein in the arterial wall is a key molecular event on the pathogenesis of atherosclerosis. We aimed to develop a NIRF-based antibody-targeted molecular imaging approach for identifying oxidised LDL (oxLDL) in vivo. Methods and results: LO1 is a naturally occurring IgG3κ autoantibody isolated from an LDL receptor deficient hypercholesterolaemic mouse. It reacts with malondialdehyde-conjugated LDL (MDA-LDL), an epitope expressed in oxidised LDL and which is recognised as reflecting vulnerability in human atherosclerotic lesions. Immunohistochemical (IHC) staining of biotinylated LO1 showed colocalisation to advanced mouse atherosclerotic plaque and to necrotic core within culprit human carotid atherosclerotic plaque. We used the immunoglobulin SH groups to successfully label LO1 with a Near Infra Red Fluorescence (NIRF) agent for in vivo imaging studies. Using two optical imaging techniques (IVIS Spectrum combined with hybrid high resolution CT as well as Fluorescence Molecular Tomography (FMT)) we demonstrated that LO1 sensitively and specifically identifies atherosclerotic lesions in vivo in the aorta of the LDLr-/- atherosclerotic mouse model but not in control mice. LO1 also demonstrated superior specificity to lesional regions of interest (ROIs) than a caged MMP probe (MMP-sense). En face immunohistochemistry on aortae from mice injected with labelled LO1 and an anti CD-31 antibody showed localisation of the targeting antibody to the sub-endothelium of the aortic arch and root. LO1 was successfully sequenced and sequence verified as germline. It was then expressed in HEK cells as a chimeric Fab (∼50kD), with murine VH and VL and human CH1 and CL. The terminus of CH1 extends to the THTC of the hinge region to provide the free cysteine for NIRF labelling. In vitro characterisation of the construct revealed maintained function following affinity purification on an anti-human CH1 column and labelling with a NIRF agent. Initial in vivo work confirms localisation to atherosclerotic lesions within the LDLr-/- model. Conclusion: LO1 and its molecularly expressed human chimeric construct (LO1-Fab-cys) provide useful tools for imaging and therapeutic targeting of oxidised LDL in atherosclerosis. Further imaging studies will be enabled by the development new technologies such as the combined Optical Fluorescence Domain Imaging and Near Infrared Fluorescence (OFDI/NIRF) intravascular catheter.

Passive Immunization with Hypochlorite-oxLDL Specific Antibodies Reduces Plaque Volume in LDL Receptor-Deficient Mice

AimsNew strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.Methods and ResultsMonoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLr−/− mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr−/− mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.ConclusionOur data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases.

GDF-15 protects from macrophage accumulation in a mousemodel of advanced atherosclerosis

BackgroundThe cytokine growth differentiation factor-15 (GDF-15), a member of the TGF beta superfamily, has recently been discovered to play an important role in cardiovascular diseases. It is mostly expressed in macrophages of atherosclerotic lesions, but its impact on advanced atherosclerosis is still unknown. This study was performed to evaluate the effects of GDF-15 in an established mouse model of advanced atherosclerosis.MethodsThirty-eight LDL receptor deficient mice received a lethal body radiation. Half of the group was transplanted with bone marrow of GDF-15 deficient mice. Nineteen mice were transplanted with bone marrow from wild-type controls. After 24 weeks on an atherogenic diet, animals were euthanized and sections of the aortic sinus were prepared. Lesion size and lesion composition, as well as macrophage content,were evaluated.ResultsWhile demonstrating no difference in lesion size, LDL-receptor knockout mice transplanted with bone marrow from GDF-15 deficient mice showed enhanced macrophage accumulation and features of atherosclerotic plaque destabilization, such as thinning of fibrous caps. Immunostaining against intercellular adhesion molecule-1 further revealed an increased expression in mice receiving GDF-15-deficient bone marrow.ConclusionsThis is the first study that demonstrates a protective role of GDF-15 in advanced atherosclerosis and macrophage accumulation, possibly due to the reduced expression of adhesion molecules.

Oxidised plant sterols as well as oxycholesterol increase the proportion of severe atherosclerotic lesions in female LDL receptor+/ −mice

Oxysterols (oxidised cholesterol) may play a role in the pathogenesis of CVD. Similar to cholesterol, plant sterols are susceptible to oxidation. However, less is known about the potential atherogenicity of oxidised plant sterols (oxyphytosterols). In the present study, the atherogenicity of a mixture of oxyphytosterols was examined by feeding female LDL receptor-deficient (LDLR+/-) mice for 35 weeks a control diet (atherogenic high-fat diet; n 9), an oxysterol diet (control diet+0·025% (w/w) oxysterols; n 12) or an oxyphytosterol diet (control diet+0·025% (w/w) oxyphytosterols; n 12). In the LDLR+/- mice, serum levels of cholesterol, lipoprotein profiles, cholesterol exposure and inflammatory markers at the end of the experiment were comparable between the three diet groups. Nevertheless, the proportion of severe atherosclerotic lesions was significantly higher after oxysterol (41%; P=0·004) and oxyphytosterol (34%; P=0·011) diet consumption than after control diet consumption (26%). Oxyphytosterol levels in the lesions were the highest in the oxyphytosterol group. Here, we show that not only dietary oxysterols but also dietary oxyphytosterols increase the proportion of severe atherosclerotic lesions. This suggests that plant sterols when oxidised may increase atherosclerotic lesion severity instead of lowering the size and severity of lesions when fed in their non-oxidised form. Therefore, this finding might give an indication as to where to find the answer in the current hot debate about the potential atherogenicity of plant sterols. However, to what extent these results can be extrapolated to the human situation warrants further investigation.