Development Of Atherosclerosis In Mice Research Articles

All omega 3 fatty acids improve serum lipid profiles and decrease aortic plaque buildup in LDLr‐/‐ mice fed an atherogenic diet (40.1)

Although the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on vascular disease markers have been well studied, the effect of docosapentaenoic acid omega‐3 (DPAn3) has been studied less. The objective of this study was to compare the effects of DPAn3, EPA, DHA, and commercial combinations of EPA, DPAn3, and DHA on serum lipid profiles and the development of atherosclerosis in mice. Male LDLr‐/‐ mice were assigned to one of six atherogenic (42% kcal from fat) dietary treatments (n=8) that had 3.5% of total fatty acids (FA) replaced with milk fat, DHA, DPAn3, EPA, or a commercial n3 fatty acid preparation. Blood samples were taken at 0, 10, and 20 weeks into the study; serum was isolated and probed for lipid classes and glucose. Aortic arches were harvested from all mice and total, free, and esterified cholesterol are reported. Serum lipid profiles and glucose in mice increased over the study, but supplementing n3 polyunsaturated fatty acids (PUFA) decreased (P<0.05) serum cholesterol, triglyceride, and non‐esterified fatty acid concentrations compared to the milk fat control. Mice supplemented with all n3 PUFA had decreased aortic total cholesterol (P<0.10) and cholesterol ester (P<0.05) compared to the control mice. The results of this study suggest that EPA, DPA, and DHA may equally (P>0.10) improve serum lipid profiles and decrease aortic plaque buildup in a mouse model prone to atherosclerosis.Grant Funding Source: Supported by Omega Protein Corporation

Comparative Genome-Wide Association Studies in Mice and Humans for Trimethylamine N -Oxide, a Proatherogenic Metabolite of Choline and l -Carnitine

Elevated levels of plasma trimethylamine N-oxide (TMAO), the product of gut microbiome and hepatic-mediated metabolism of dietary choline and L-carnitine, have recently been identified as a novel risk factor for the development of atherosclerosis in mice and humans. The goal of this study was to identify the genetic factors associated with plasma TMAO levels. We used comparative genome-wide association study approaches to discover loci for plasma TMAO levels in mice and humans. A genome-wide association study in the hybrid mouse diversity panel identified a locus for TMAO levels on chromosome 3 (P=2.37 × 10(-6)) that colocalized with a highly significant (P=1.07 × 10(-20)) cis-expression quantitative trait locus for solute carrier family 30 member 7. This zinc transporter could thus represent 1 positional candidate gene responsible for the association signal at this locus in mice. A genome-wide association study for plasma TMAO levels in 1973 humans identified 2 loci with suggestive evidence of association (P=3.0 × 10(-7)) on chromosomes 1q23.3 and 2p12. However, genotyping of the lead variants at these loci in 1892 additional subjects failed to replicate their association with plasma TMAO levels. The results of these limited observational studies indicate that, at least in humans, genes play a marginal role in determining TMAO levels and that any genetic effects are relatively weak and complex. Variation in diet or the repertoire of gut microbiota may be more important determinants of plasma TMAO levels in mice and humans, which should be investigated in future studies.

Targeted mouse complement inhibitor CR2-Crry protects against the development of atherosclerosis in mice

ObjectiveAtherosclerosis is a chronic inflammatory and immune vascular disease, and clinical and experimental evidence has indicated an important role of complement activation products, including the terminal membrane attack complex (MAC), in atherogenesis. Here, we investigated whether complement inhibition represents a potential therapeutic strategy to treat/prevent atherogenesis using CR2-Crry, a recently described complement inhibitor that specifically targets to sites of C3 activation. Methods and resultsPrevious studies demonstrated that loss of CD59 (a membrane inhibitor of MAC formation) accelerated atherogenesis in Apoe deficient (Apoe−/−) mice. Here, both CD59 sufficient and CD59 deficient mice in an Apoe deficient background (namely, mCd59 ab+/+/Apoe−/− and mCd59 ab−/−/Apoe−/−) were treated with CR2-Crry for 4 and 2 months respectively, while maintained on a high fat diet. Compared to control treatment, CR2-Crry treatment resulted in significantly fewer atherosclerotic lesions in the aorta and aortic root, and inhibited the accelerated atherogenesis seen in mCd59 ab+/+/Apoe−/− and mCd59 ab−/−/Apoe−/− mice. CR2-Crry treatment also resulted in significantly reduced C3 and MAC deposition in the vasculature of both mice, as well as a significant reduction in the number of infiltrating macrophages and T cells. ConclusionThe data demonstrate the therapeutic potential of targeted complement inhibition.

Abstract 19061: Kca3.1 Regulates Monocytosis and Monocyte Adherence in Atherosclerosis

Introduction: Atherosclerosis is associated with a monocytosis of an inflammatory subset (Ly-6Chi in mice), which is preferentially recruited to lesions and gives rise to pro-atherosclerotic macrophages (MΦ). We have shown that the intermediate-conductance Ca2+-activated K+ channel KCa3.1 is predominantly expressed in blood Ly-6Chi monocytes (MONO) and lesion MΦ, and KCa3.1 blockade or knockout reduces the development of atherosclerosis in mice. To further study the function of KCa3.1 in atherogenesis, we investigated its role in monocytosis and in MONO adherence to the endothelium in vivo. Methods and Results: Flow cytometric analyses of peripheral blood and bone marrow from Apoe-/- and Apoe-/- KCa3.1-/- mice showed that KCa3.1 deficiency decreased circulating MONO (8.8±1.5% of leukocytes, p<0.05 vs. 15.4±2.1%; n=7-8) and Ly6Chi MONO (4.2±0.7% of leukocytes, p<0.05 vs. 9.8±1.5%, n=7-8), with unchanged Ly6Clo MONO (4.6±1.3% of leukocytes, p=ns vs. 5.6±0.7%) and polymorphonuclar (11.7±1.4% of cells, p=ns vs. 12.5±1.2%; n=7-8). Apoe-/- KCa3.1-/- mice had reduced hematopoietic stem and multipotential progenitor cells (HSPCs) (2.4±0.5% of lineage negative cells, p<0.05 vs. 3.0±0.5%, n=7). In vivo confocal endomicroscopic analysis of leukocyte adherence was performed in anesthetized mice 4 hours after topical application of 0.5 μg TNF-α and 0.125 μg IL-1β onto the right external carotid artery where an imaging probe (Cellvizio®, field of view=8x105 μm2) was placed. Blood and circulating leukocytes were stained by intravenous injection of 200 μg FITC-dextran and 0.02% acrydine orange, respectively. KCa3.1 deficiency markedly inhibited leukocyte rolling (4±2 vs. 52±15 cells/min, contact time: 1.6±0.5 vs. 2.7±0.2 sec, distance: 78±11 vs. 165±5 μm, and velocity: 145±24 vs. 119±5 μm/sec) and adhesion (0.5±0.3 vs. 2.0±0.6 cells/min, p<0.05 for all data, n=6). Immunohistochemistry of lesions showed that Apoe-/- KCa3.1-/- mice had less CD68-positive MONO/MΦ compared to Apoe-/- mice (616±101, p<0.05 vs. 937±90 cells/mm2, n=8-9). Conclusion: KCa3.1 modulates monocytosis and plays a major role in MONO adherence to the endothelium in vivo. Thus KCa3.1 constitutes a promising therapeutic target for the prevention of atherosclerosis.

Interleukin 15 Plays an Important Role in the Development of Atherosclerosis in Mice

IL-15 is an essential cytokine for the development of NK and CD8+T cells and also involved in activation of inflammation in macrophages. Therefore, we hypothesized that IL-15 may affect the development of atherosclerosis via regulating immune/inflammatory cells. To test this we generated apoE KO mice that either overexpressed (il-15TgapoE-/-) or lacked either one (il-15+/-apoE-/-) or both copies (il-15-/-apoE-/-) of the IL-15 gene. Deficiency of IL-15 substantially reduced atherosclerosis in spite of increased plasma lipoprotein cholesterol levels.

Comparison of Oral Tolerance to ApoB and HSP60 Peptides in Preventing Atherosclerosis Lesion Formation in Apob48−/Ldlr− Mice

Antigen-specific immune modulation is emerging as an attractive therapeutic option to prevent atherosclerosis. We compared the efficacy of oral administration of peptides derived from apolipoprotein B (ApoB; 661–680) and heat shock protein 60 (HSP60; 153–163), in the prevention of atherosclerotic lesion formation hyperlipidemic low density lipoprotein receptordeficient (LDLr−/−), apolipoprotein B-100 only (apoB100/100) mice model. Oral administration of peptides induced tolerance as seen by an increase in regulatory T cells in the peripheral immune system. Tolerance to ApoB peptide reduced plaque development by 28.7% (P<0.001) while HSP60 was effective in reducing lesion development by 26.8% in ApoB48/LDLr−/− mice. While tolerance to HSP60 resulted in increase in anti-inflammatory cytokines (IL10 and TGF-β), ApoB tolerance was effective in reducing the lipid deposition in the lesion. Our results suggest that the two peptides have distinct mechanisms of controlling the development of atherosclerosis in mice.

Bone marrow NR4A expression is not a dominant factor in the development of atherosclerosis or macrophage polarization in mice

The formation of the atherosclerotic lesion is a complex process influenced by an array of inflammatory and lipid metabolism pathways. We previously demonstrated that NR4A nuclear receptors are highly induced in macrophages in response to inflammatory stimuli and modulate the expression of genes linked to inflammation in vitro. Here we used mouse genetic models to assess the impact of NR4A expression on atherosclerosis development and macrophage polarization. Transplantation of wild-type, Nur77−/−, or Nor1−/− null hematopoetic precursors into LDL receptor (LDLR)−/− recipient mice led to comparable development of atherosclerotic lesions after high-cholesterol diet. We also observed comparable induction of genes linked to M1 and M2 responses in wild-type and Nur77-null macrophages in response to lipopolysaccharides and interleukin (IL)-4, respectively. In contrast, activation of the nuclear receptor liver X receptor (LXR) strongly suppressed M1 responses, and ablation of signal transductor and activator of transcription 6 (STAT6) strongly suppressed M2 responses. Recent studies have suggested that alterations in levels of Ly6Clo monocytes may be a contributor to inflammation and atherosclerosis. In our study, loss of Nur77, but not Nor1, was associated with decreased abundance of Ly6Clo monocytes, but this change was not correlated with atherosclerotic lesion development. Collectively, our results suggest that alterations in the Ly6Clo monocyte population and bone marrow NR4A expression do not play dominant roles in macrophage polarization or the development of atherosclerosis in mice.

Prenatal stress enhances severity of atherosclerosis in the adult apolipoprotein E-deficient mouse offspring via inflammatory pathways.

Atherosclerosis is the underlying cause of cardiovascular disease and stroke. Endothelial cell dysfunctions are early events in atherosclerosis, resulting in the recruitment of circulating monocytes. The immune system can elicit an inflammatory response toward the atherosclerotic lesion, thereby accelerating lesion growth. Risk factors for atherosclerosis include hypertension, smoking, stress perception or low birth weight. As prenatal stress challenge decreases the birth weight and affects the offspring's postnatal immune response, we aimed to investigate whether prenatal stress contributes to the development of atherosclerosis in mice. Syngenic pregnant apolipoprotein E-deficient (apoE-/-) dams were exposed to sound stress on gestation days 12.5 and 14.5. The presence and size of atherosclerotic plaques in the offspring at the age of 15 weeks was evaluated by histomorphology, accompanied by flow cytometric analysis of the frequency and phenotype of monocytes/macrophages and regulatory T (Treg) cells in the blood. Further, cytokine secretion of peripheral blood lymphocytes was analyzed. In response to prenatal stress challenge, an increased frequency of large atherosclerotic plaques was detectable in apoE-/- offspring, which was particularly profound in females. Prenatal stress also resulted in alterations of the offspring's immune response, such as a decreased frequency of Treg cells in blood, alterations of macrophage populations in blood and an increased secretion of inflammatory cytokines. We provide novel evidence that prenatally stressed adult offspring show an increased severity of atherosclerosis. As Treg cells are key players in dampening inflammation, the observed increase in atherosclerosis may be due to the lack of Treg cell frequency. Future interdisciplinary research is urgently required to understand the developmental origin of prenatal stress-induced atherosclerosis. The availability of our model may facilitate and foster such research endeavors.

Mechanisms and dose–response relationships for radiation-induced cardiovascular disease

Epidemiological studies have shown a clear association between therapeutic doses of thoracic irradiation and increased risk of cardiovascular disease in long-term cancer survivors. Survivors of Hodgkin’s lymphoma and childhood cancers, for example, show 2- to >7-fold increases in risk of cardiac death after total tumour doses of 30–40Gy, given in 2-Gy fractions. The risk of cardiac mortality increases linearly with dose, although there are large uncertainties for mean cardiac doses <5Gy. Experimental studies show that doses of ⩾2Gy induce the expression of inflammatory and thrombotic molecules in endothelial cells. In the heart, this causes progressive loss of capillaries and eventually leads to reduced perfusion, myocardial cell death, and fibrosis. In large arteries, doses of ⩾8Gy, combined with elevated cholesterol, initiates atherosclerosis and predisposes to the formation of inflammatory, unstable lesions, which are prone to rupture and may cause a fatal heart attack or stroke. In contrast, doses <1Gy inhibit inflammatory cell adhesion to endothelial cells and inhibit the development of atherosclerosis in mice. It seems likely that mechanisms other than accelerated atherosclerosis are responsible for cardiovascular effects after low total-body exposures of radiation (e.g. impaired T-cell immunity or persistent increase in systemic cytokines).

Macrophage paraoxonase 2 regulates calcium homeostasis and cell survival under endoplasmic reticulum stress conditions and is sufficient to prevent the development of aggravated atherosclerosis in paraoxonase 2 deficiency/apoE−/− mice on a Western diet

Paraoxonase 2 deficiency (PON2-def) alters mitochondrial function and exacerbates the development of atherosclerosis in mice. PON2 overexpression protects against ER stress in cell culture. In this paper, we examined the role of PON2 in the unexplored link between ER stress and mitochondrial dysfunction and tested whether restoration of PON2 in macrophages is sufficient to reduce aggravated atherosclerosis in PON2-def/apoE−/− mice on a Western diet. ER stress response genes, intracellular calcium levels, and apoptotic nuclei were significantly elevated in PON2-def/apoE−/− macrophages compared to apoE−/− macrophages in response to ER stressors, but not at the basal level. In contrast, PON2-def/apoE−/− macrophages exhibited greater mitochondrial stress at the basal level, which was further worsened in response to ER stressors. There was no difference in ER stress response genes and apoptotic nuclei between apoE−/− and PON2-def/apoE−/− macrophages when pretreated with xestospongin (which blocks the release of calcium from ER) suggesting that PON2 modulates cell survival and ER stress by maintaining calcium homeostasis. Treatment with a mitochondrial calcium uptake inhibitor, RU360, attenuated ER stressor mediated mitochondrial dysfunction in PON2-def/apoE−/− macrophages. CHOP expression (ER stress marker) and apoptotic nuclei were significantly higher in aortic lesions of PON2-def/apoE−/− mice compared to apoE−/− mice fed a Western diet. Restoration of PON2 in macrophages reduced ER stress, mitochondrial dysfunction and apoptosis in response to ER stressors. Furthermore, restoration of PON2 in macrophages reduced lesional apoptosis and atherosclerosis in PON2-def/apoE−/− mice on a Western diet. Our data suggest that macrophage PON2 modulates mechanisms that link ER stress, mitochondrial dysfunction and the development of atherosclerosis.

Bmper Inhibits Endothelial Expression of Inflammatory Adhesion Molecules and Protects Against Atherosclerosis

Bone morphogenetic proteins (Bmps) are important mediators of inflammation and atherosclerosis, though their mechanism of action is not fully understood. To better understand the contribution of the Bmp signaling pathway in vascular inflammation, we investigated the role of Bmper (Bmp endothelial cell precursor-derived regulator), an extracellular Bmp modulator, in an induced in vivo model of inflammation and atherosclerosis. We crossed apolipoprotein E-deficient (ApoE(-/-)) mice with mice missing 1 allele of Bmper (Bmper(+/-) mice used in the place of Bmper(-/-) mice that die at birth) and measured the development of atherosclerosis in mice fed a high-fat diet. Bmper haploinsufficiency in ApoE(-/-) mice (Bmper(+/-);ApoE(-/-) mice) led to a more severe phenotype compared with Bmper(+/+);ApoE(-/-) mice. Bmper(+/-);ApoE(-/-) mice also exhibited increased Bmp activity in the endothelial cells in both the greater and lesser curvatures of the aortic arch, suggesting a role for Bmper in regulating Bmp-mediated inflammation associated with laminar and oscillatory shear stress. Small interfering RNA knockdown of Bmper in human umbilical vein endothelial cells caused a dramatic increase in the inflammatory markers intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 at rest and after exposure to oscillatory and laminar shear stress. We conclude that Bmper is a critical regulator of Bmp-mediated vascular inflammation and that the fine-tuning of Bmp and Bmper levels is essential in the maintenance of normal vascular homeostasis.

Abstract 43: The Metalloprotease ADAMTS13 Reduces VWF-Mediated Vascular Inflammation and Early Development of Atherosclerosis in Mice

Background and objective: ADAMTS13 prevents spontaneous thrombosis in the microvasculature by cleaving hyperactive ultra large von Willebrand factor (ULVWF) multimers into smaller and less active forms. Reduced ADAMTS13 activity and increased VWF levels have been described in many diseases associated with systemic inflammation. We hypothesized that, by cleaving ULVWF multimers and/or VWF multimers, ADAMTS13 reduces vascular inflammation and the development of early atherosclerotic plaques. Model and methods: ApoE -/-, Adamts13-/-/ApoE-/-, Adamts13 -/-/ Vwf -/-/ ApoE -/- and Vwf -/-/ApoE -/- mice were fed a high-fat Western diet (20% fat, 0.2% cholesterol) beginning at 6 weeks of age until they were sacrificed at 4 months. Intravital fluorescence microscopy was used to visualize leukocyte adhesion and plaque formation at the carotid sinus. We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain. Inflammatory cells in the aortic lesions were quantitated by immunohistochemistry. Results: Using intravital microscopy, we observed a 4-fold increase ( P &lt;0.001) in leukocyte adhesion and at the carotid sinus of Adamts13-/-/ApoE -/- mice compared with ApoE -/- mice. Interestingly, 100% of the Adamts13-/-/ApoE -/- mice had larger plaque (occluded vessel by ∼70-80 %) at the carotid sinus whereas only 20% of the ApoE- /- mice had plaques that were smaller in size ( P &lt;0.0001). Next, we found that the atherosclerotic lesions in the aorta and aortic sinus of the Adamts13-/-/ApoE-/- mice were not only larger (2-fold, P &lt;0.001) but also more inflammatory (increased neutrophil and macrophage infiltration) with decreased interstitial collagen compared with ApoE -/- mice, suggesting that ADAMTS13 reduces vascular inflammation and subsequent atherosclerotic plaque formation. Adamts13-/-/ApoE -/- mice fed a normal chow diet also demonstrated significantly accelerated atherosclerotic plaque formation compared with ApoE -/- mice. Total cholesterol and triglyceride levels were similar among groups fed high-fat Western or normal chow diets. Finally, atherosclerotic lesions in Adamts13-/-/Vwf-/-/ApoE -/- mice were similar to Vwf-/-/ApoE -/- mice, demonstrating that the accelerated atherosclerosis observed in ADAMTS13-deficient mice is VWF-dependent. Conclusion: These findings reveal a new role for the antithrombotic enzyme ADAMTS13 in reducing VWF-mediated plaque formation during early atherosclerosis.

Roles of TRAF5 in TLR signaling (180.4)

Abstract Dysregulation in Toll-like receptor (TLR) signaling has been implicated in many disease states, including chronic inflammatory conditions such as atherosclerosis. The cytoplasmic adaptor proteins TNF receptor associated factor (TRAF)3 and TRAF6 are known to mediate TLR signaling. Recent data from our lab demonstrate for the first time that TRAF5 also plays a significant role in immune cell TLR functions. B lymphocytes, macrophages, and dendritic cells from TRAF5-/- mice show increased cytokine production and enhanced early signaling pathways after stimulation through various TLRs. Interestingly, B cell TLR responses are the most greatly affected by TRAF5 deficiency. Our data show that TRAF5 is a negative regulator of TLR signaling and restrains proinflammatory cytokine production. It has been reported that TRAF5 deficiency accelerates the development of atherosclerosis in mice, while human patients with coronary heart disease express lower levels of TRAF5 mRNA in blood compared with healthy controls. Elucidating the role of TRAF5 in TLR signaling may provide a new target for therapies against a variety of inflammatory conditions, including those predisposing to atherogenesis. Our current studies focus on identifying the key molecular interactions by which TRAF5 mediates its effects on TLR pathways.

Oral FTY720 Administration Induces Immune Tolerance and Inhibits Early Development of Atherosclerosis in Apolipoprotein E-Deficient Mice

Orally administered immunomodulatory drugs have recently demonstrated the ability to induce an oral tolerance via inhibition of effector T cells and induction of certain subsets of regulatory T cells (Tregs) which have the potential to prevent several autoimmune diseases. In the present study, we hypothesized that short-term, low-dose, oral FTY720 administration may induce latency-associated peptide (LAP) Tregs and CD4(+) Foxp3(+) Tregs in atherogenesis, potentially resulting in remission of early development of atherosclerosis in apolipoprotein E-deficient (APOE(-/-)) mice. FTY720 was orally administered to APOE(-/-) mice 4 weeks of age on a high-cholesterol diet and atherosclerosis was assessed at 8 weeks of age. Oral administration of FTY720 significantly reduced atherosclerotic lesion formation compared with control mice. We observed a significant increase in LAP(+) and Foxp3(+) cells in the CD4+T-cell population of FTY720-treated mice in association with increased production of the anti-inflammatory cytokine transforming growth factor-β (TGF-β) as well as suppressed T-helper type 1 immune responses. Our findings reveal that short-term, low-dose oral FTY720 treatment had great benefits in inhibiting early development of atherosclerosis in mice via induction of a regulatory T-cell response and inhibition of effector T responses. These findings suggest that oral immune modulation may represent an attractive therapeutic approach to atherosclerosis.

Abstract P141: Gender Differences in the Development of Severe Pulmonary Hypertension and Right Ventricular Dysfunction in Apolipoprotein E--Deficient Mice Are Eliminated with Increasing Age

Apolipoprotein E (ApoE) is a multifunctional protein and its deficiency leads to the development of atherosclerosis in mice. Patients with pulmonary hypertension (PH) have reduced expression of ApoE in lung tissue. ApoE is known to inhibit endothelial and smooth muscle cell proliferation and has anti-inflammatory and anti-platelet aggregation properties. Young ApoE deficient mice have been shown to develop high fat diet-induced PH in a gender specific manner. Estrous cyclicity peaks at 7–8 months and declines by 9 months of age in mice. Here we investigated the effects of monocrotaline (MCT) on young and middle-aged ApoE deficient mice.Middle-Aged (MA) (11–12 month old) male (n=4) and female (n=4) and young (7–8 month old) male (n=5) and female (n=5) ApoE deficient mice were injected with a single intraperitoneal dose of MCT (60 mg/kg). Mice were closely monitored for ∼4 weeks with serial echocardiography for cardiopulmonary hemodynamic assessment. Direct cardiac catheterisation was performed terminally to record peak systolic right ventricular pressure (RVP). RV, LV, IVS and lung tissue was dissected and weighed. Trichrome staining and histochemical analyses were performed. At ∼4 weeks after MCT, MA male and female and young male mice developed severe PH (RVP: MA male=64±5 mmHg, MA female=71±4 mmHg, young male=60±5 mmHg, p=n.s between all the groups) whereas young females developed significantly less severe PH (RVP: 37±5 mmHg, P&lt;0.05 vs. MA male and female, and young male). MA male and female and young male mice developed severe RV dysfunction (RV ejection fraction (RVEF): MA male=31±2%, MA female=28±4%, young male=36±1%, p=n.s between all the groups) whereas young females showed significantly better RV function (RVEF: 43±2%, P&lt;0.05 vs. MA male and female, and young male). MA male and female mice also developed more severe RV hypertrophy (RV/LV+Septum, MA male=0.49, MA female=0.53, young female=0.39). MA male and female mice also manifested increased peripheral pulmonary artery muscularization and pulmonary fibrosis. Interestingly, the gender differences witnessed between young ApoE deficient male and female mice in the development of severe PH and RV dysfunction are abolished as the mice increase in age.

Fibronectin Isoform Containing the Alternatively-Spliced Extra Domain A in Circulation Accelerates the Development of Atherosclerosis in Mice

Abstract 2205 Background and Objective:The fibronectin isoform containing the alternatively-spliced extra domain A (EDA+-FN) is normally absent from the circulation, but plasma levels of EDA+-FN can become markedly elevated in several pathological conditions including atherosclerosis. It remains unclear in humans whether these elevated levels of EDA+-FN are actively contributing to disease pathogenesis, or rather simply serving as a marker associated with vascular stress and/or injury. Several in vitro studies suggest that EDA+-FN can activate toll-like receptor 4 (TLR4), an innate immune receptor that triggers pro-inflammatory responses We hypothesize that presence of EDA+-FN in plasma promotes inflammation and accelerates atherosclerotic plaque formation. Model and Method:We generated EDA+/+/ApoE−/− mice, which contain optimized spliced sites at both splicing junctions of the EDA exon and constitutively express only EDA+-FN, and EDA−/−/ApoE−/− mice, which contain an EDA-null allele of the EDA exon and express only FN lacking EDA. ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain. Results:We report that atherosclerotic plaque (% of total aorta) formation in the aorta of EDA+/+/ApoE−/− mice was increased by two-fold compared to control ApoE−/− mice (P<0.0001). Deletion of the alternatively spliced EDA domain in the ApoE−/− mice (EDA−/−/ApoE−/−) significantly reduced atherosclerotic plaque formation in the aorta (P<0.05) compared to ApoE−/− mice. Total cholesterol and triglycerides levels were similar in ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− mice. Similarly, atherosclerotic plaque formation was significantly increased in the aortic sinus of EDA+/+/ApoE−/− mice, intermediate in control ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice (P<0.05). Additionally, we found that macrophage content, as analyzed by immunohistochemistry, was significantly elevated in the aortic root lesions of EDA+/+/ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice compared to ApoE−/− mice (P<0.05). Moreover, EDA+-FN did not affect the sex-dependent regulation of atherosclerosis in ApoE−/− mice. Future experiments using EDA+/+/ApoE−/−/TLR4−/− are under progress to determine whether EDA+-FN exacerbate atherosclerosis via upregulating TLR4 signaling. Conclusions:Our findings reveal that EDA+-FN is pro-inflammatory and promotes atherosclerotic lesion formation and that monitoring plasma EDA+-FN might have prognostic value in patients at high risk for atherosclerosis. Disclosures:No relevant conflicts of interest to declare.

The Anti-Thrombotic Enzyme ADAMTS13 Reduces Inflammation and the Development of Atherosclerosis In Mice

Abstract 367 Background and objective:ADAMTS13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13) plays a pivotal role in preventing spontaneous thrombosis in the microvasculature by cleaving hyperactive ultra large von Willebrand factor (ULVWF) multimers into smaller, less active multimers. Severe deficiency of ADAMTS13 in humans causes thrombotic thrombocytopenic purpura (TTP) and numerous epidemiological studies have demonstrated associations between decreased ADAMTS13 activity and adverse disease outcome in patients with systemic inflammation. It remains unknown, however, whether reduced ADAMTS13 activity plays a direct pathogenic role in inflammatory diseases or rather simply serves as an inflammation-associated marker. We hypothesized that deficiency of ADAMTS13 enhances inflammation and accelerates the development of early atherosclerotic plaques. Results:Using intravital fluorescence microscopy, we show that the number of adherent leukocytes (adherent for > 60 s) was increased approximately four-fold at the carotid sinus, a lesion prone site, of Adamts13−/−/ApoE−/− mice (Mean ± SEM = 37 ± 6) as compared to ApoE−/− mice (Mean ± SEM = 9 ± 4, P <0.01) fed a high-fat Western diet. Interestingly, intravital microscopy showed that 100% (10/10) of the Adamts13−/−/ApoE−/− mice had plaque that occluded the carotid sinus by approximately 70–80%, whereas only 20% (2/10) of the ApoE−/− mice had plaque at the carotid sinus, and the plaques were smaller in size than those in Adamts13−/−/ApoE−/− mice (P=0.0003). Next, we determined the effects of ADAMTS13 deficiency on atherosclerotic plaque formation in the aorta and aortic sinus. We compared the extent of atherosclerosis in whole aortae stained with Oil Red O and en face lesion area measured by morphometry. Both Adamts13−/−/ApoE−/− male and female mice demonstrated significantly larger lesions in the descending aorta (P<0.01), arch of the aorta (P<0.001), and total aorta (P<0.0001) compared to ApoE−/− mice fed a high-fat Western diet. Next, we quantified the corss-sectional area of lesions in the aortic sinus using the VerHoeffs/Van Gieson method. We observed a two-fold increase in the mean lesion area in the aortic sinus of both male and female Adamts13−/−/ApoE−/− mice (P<0.01) compared to ApoE−/− mice. Macrophage content (% of total lesion area), as quantitated by immunohistochemistry, was significantly elevated in the aortic root lesions of Adamts13−/−/ApoE−/− mice compared to ApoE−/− mice, suggesting that exacerbated atherosclerosis was due to increased inflammation. Adamts13−/−/ApoE−/− mice fed a normal chow diet also demonstrated accelerated atherosclerotic plaque formation compared to ApoE−/− mice. Total cholesterol and triglyceride levels were similar in Adamts13−/−/ApoE−/− and ApoE−/− mice fed a high-fat Western diet or normal chow diet. Conclusions:These findings unravel a new functional role for the anti-thrombotic enzyme ADAMTS13 in reducing excessive inflammation and plaque formation during atherosclerosis. Disclosures:Lentz:Celgene: Ownership interest; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Low-Density Lipoprotein Receptor–Related Protein 1 Prevents Early Atherosclerosis by Limiting Lesional Apoptosis and Inflammatory Ly-6C high Monocytosis

Background— We previously demonstrated that macrophage low-density lipoprotein receptor (LDLR)–related protein 1 (LRP1) deficiency increases atherosclerosis despite antiatherogenic changes including decreased uptake of remnants and increased secretion of apolipoprotein E (apoE). Thus, our objective was to determine whether the atheroprotective effects of LRP1 require interaction with apoE, one of its ligands with multiple beneficial effects. Methods and Results— We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE −/− MΦLRP1 −/− ) and in LDLR −/− mice reconstituted with apoE −/− MΦLRP1 −/− bone marrow. The combined absence of apoE and LRP1 promoted atherogenesis more than did macrophage apoE deletion alone in both apoE-producing LDLR −/− mice (+88%) and apoE −/− mice (+163%). The lesions of both mouse models with apoE −/− LRP1 −/− macrophages had increased macrophage content. In vitro, apoE and LRP1 additively inhibit macrophage apoptosis. Furthermore, there was excessive accumulation of apoptotic cells in lesions of both LDLR −/− mice (+110%) and apoE −/− MΦLRP1 −/− mice (+252%). The apoptotic cell accumulation was partially due to decreased efferocytosis as the ratio of free to cell-associated apoptotic nuclei was 3.5-fold higher in lesions of apoE −/− MΦLRP1 −/− versus apoE −/− mice. Lesion necrosis was also increased (6 fold) in apoE −/− MΦLRP1 −/− versus apoE −/− mice. Compared with apoE −/− mice, the spleens of apoE −/− MΦLRP1 −/− mice contained 1.6- and 2.4-fold more total and Ly6-C high monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-C high and CC-chemokine receptor 2–positive cells in lesions of apoE −/− MΦLRP1 −/− versus apoE −/− mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes. Conclusion— Low-density lipoprotein receptor protein 1 exerts antiatherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.

Curcumin inhibits atherosclerosis by suppressing accumulation of lipids in macrophages in LDLr −/− mice

Atherosclerosis is a multi-factorial disease of arteries where macrophages accumulate lipids and cholesterol through scavenger receptors and cytosolic fatty acid binding proteins (FABPs) leading to formation of foam cells. Knockout of FABP-4 or (aP2) gene attenuated the development of atherosclerosis in mice. Earlier we found that curcumin, a polyphenol from turmeric spice, down-regulated expression of aP2 in 3T3L-1 adipocytes and THP-1 macrophages and reduced accumulation of lipids in these cells in vitro. We hypothesized that dietary supplementation of curcumin in LDLr−/− mice may attenuate high fat diet–induced atherogenesis by suppressing aP2 expression, accumulation of lipids in macrophage and inflammatory cytokines. 8-wk old LDLr−/− mice were either fed control or high fat (HF; 20% w/w) diet supplemented with curcumin (0, 500, 1000, 1500 mg/kg diet) for 16 wks (n=24/group). Dietary curcumin supplementation had no effect on food intake and intestinal fat absorption, but significantly attenuated body wt. gain and adiposity (MRI) in HF-fed mice. Curcumin dose-dependently reduced accumulation of lipids in peritoneal macrophages of HF-fed mice. Analysis of atherosclerotic lesions in lumen of aortic tree revealed that dietary curcumin at 500 and 1000mg/kg diet significantly attenuated atherogenesis in HF-fed mice. Grant Funding Source: USDA Award 2010-65200-203945

Secreted phospholipase A2, lipoprotein hydrolysis, and atherosclerosis: integration with lipidomics

Phospholipase A2 (PLA2) is a group of enzymes that hydrolyze the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Of many PLA2s or related enzymes identified to date, secreted PLA2s (sPLA2s) comprise the largest family that contains 10 catalytically active isozymes. Besides arachidonic acid released from cellular membranes for eicosanoid synthesis, several if not all sPLA2s have recently been implicated in hydrolysis of phospholipids in lipoprotein particles. The sPLA2-processed low-density lipoprotein (LDL) particles contain a large amount of lysophospholipids and exhibit the property of “small-dense” or “modified” LDL, which facilitates foam cell formation from macrophages. Transgenic overexpression of these sPLA2s leads to development of atherosclerosis in mice. More importantly, genetic deletion or pharmacological inhibition of particular sPLA2s significantly attenuates atherosclerosis and aneurysm. In this article, we will give an overview of current understanding of the role of sPLA2s in atherosclerosis, with recent lipidomics data showing the action of a subset of sPLA2s on lipoprotein phospholipids.