apoE-null Mice Research Articles

Imaging RAGE expression in atherosclerotic plaques in hyperlipidemic pigs.

BackgroundReceptor for advanced glycated end product (RAGE) expression is a prominent feature of atherosclerosis. We have previously shown in apoE null mice uptake of a radiolabeled anti-RAGE antibody in atherosclerotic plaque and now evaluate RAGE-directed imaging to identify advanced plaques in a large animal model.MethodsNine hyperlipidemic (HL) pigs were injected with 603.1 ± 129.5 MBq of 99mTc-anti-RAGE F(ab′)2, and after 6 h (blood pool clearance), they underwent single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of the neck, thorax, and hind limbs. Two HL pigs received 99mTc non-immune IgG F(ab′)2, and three farm pigs were injected with 99mTc-anti-RAGE F(ab′)2. After imaging, the pigs were euthanized. The aorta from the root to bifurcation was dissected, and the innominates, proximal carotids, and coronaries were dissected and counted, stained for H&E and RAGE, and AHA-classified.ResultsOn pathology, 24% of the arterial segments showed AHA class III or IV lesions, and these lesions were confined almost exclusively to coronaries and carotids with % stenosis from 15% to 65%. Scatter plots of %ID/g for class III/IV vs. I/II lesions showed almost complete separation. Focal vascular uptake of tracer visualized on SPECT scans corresponded to class III/IV lesions in the coronary and carotid vessels. In addition, uptake in the hind limbs was noted in the HL pigs and corresponded to RAGE staining of small arteries in the muscle sections. Correlations for the vascular lesions were r = 0.747, P = 0.001 for %ID vs. %ID/g and r = 0.83, P = 0.002 for %ID/g vs. % RAGE staining.ConclusionsUptake of radiolabeled anti-RAGE antibody in coronary and carotid fibroatheroma and in the small arteries of the hind limbs in a relevant large animal model of atherosclerosis supports the important role of RAGE in atherosclerosis and peripheral artery disease as a target for imaging and treatment.

171 Deleting TH1-lymphocytes Influences Macrophage Activation and Metalloproteinase Expression in apoe Null Mice

IntroductionAtherosclerosis is a chronic inflammatory disease, in which foam cell macrophage (FCM) accumulation is initiated by hypercholesterolemia. Classical (M1) and alternative (M2) activation of FCMs could play important roles in...

Active invasion of oral and aortic tissues by Porphyromonas gingivalis in mice causally links periodontitis and atherosclerosis.

Atherosclerotic vascular disease is a leading cause of myocardial infarction and cerebrovascular accident, and independent associations with periodontal disease (PD) are reported. PD is caused by polymicrobial infections and aggressive immune responses. Genomic DNA of Porphyromonas gingivalis, the best-studied bacterial pathogen associated with severe PD, is detected within atherosclerotic plaque. We examined causal relationships between chronic P. gingivalis oral infection, PD, and atherosclerosis in hyperlipidemic ApoEnull mice. ApoEnull mice (n = 24) were orally infected with P. gingivalis for 12 and 24 weeks. PD was assessed by standard clinical measurements while the aorta was examined for atherosclerotic lesions and inflammatory markers by array. Systemic inflammatory markers serum amyloid A, nitric oxide, and oxidized low-density lipoprotein were analyzed. P. gingivalis infection elicited specific antibodies and alveolar bone loss. Fluorescent in situ hybridization detected viable P. gingivalis within oral epithelium and aorta, and genomic DNA was detected within systemic organs. Aortic plaque area was significantly increased in P. gingivalis-infected mice at 24 weeks (P<0.01). Aortic RNA and protein arrays indicated a strong Th2 response. Chronic oral infection with P. gingivalis results in a specific immune response, significant increases in oral bone resorption, aortic inflammation, viable bacteria in oral epithelium and aorta, and plaque development.

Abstract 522: Shotgun Proteomics Implicates Matrix Proteolysis, Cell Adhesion and Cell Death in Urokinase-Accelerated Vascular Disease

Introduction: Elevated activity of urokinase plasminogen activator (uPA) in human arteries associates with atherosclerosis, aneurysms, and plaque rupture. However, the underlying mechanisms remain unknown. ApoE null mice with macrophage-specific uPA overexpression (SR-uPA mice) have elevated vascular uPA activity, accelerated atherosclerosis, dilated aortas, and histologic features of plaque rupture. Thus, SR-uPA mice may be a useful model of uPA-accelerated human vascular disease. Hypothesis: We hypothesized that mechanisms of uPA-accelerated vascular disease would be revealed by shotgun proteomics of aortas of SR-uPA and nontransgenic (ntg) ApoE null mice. We focused our studies on extracellular matrix (ECM) because previous studies implicate uPA in alteration of matrix-associated proteins. Methods: Using aortas, we developed a method for generating tissue extracts (TE) that selectively isolate ECM proteins. We extracted proteins from aortic arches of SR-uPA and ntg ApoE null mice. We also collected conditioned medium (CM) from their explanted aortas. Results: Mass spectrometry-based proteomics demonstrated that aortic TE were greatly enriched in ECM proteins as determined by Gene Ontology (GO) analysis. These TE contained all major and several minor vascular collagens, and many proteoglycans. CM contained a similar number of ECM proteins as TE; however, TE were enriched in collagens, proteoglycans, elastin, and laminins. Using stringent dual statistical criteria and a FDR of 5%, 107 of 1125 proteins were differentially present in SR-uPA aorta. MMP-2, -3, and -10 were increased; laminins, collagen I, fibronectin, and elastin were decreased. CM analysis revealed 1161 proteins; 57 were differentially present in SR-uPA. NuMa1, nuclear lamins, histones, actin, and myosins were increased; PAI-1, Plg, and A2m were decreased. Unbiased GO analysis helped identify proteolysis, ECM, cell adhesion, and cell death as processes that differed significantly between SR-uPA and ntg aortas. Conclusions: Elevated aortic uPA activity alters abundance of a limited number of proteins and affects specific biological processes, implicating ECM proteolysis, altered cell adhesion, and cell death in the pathogenesis of uPA-mediated vascular disease.

Improvement of HDL and atherosclerosis by a mimetic peptide (18A) complexed with niclosamide and administered orally to mice (LB532)

18A was incubated together with niclosamide and within 6h of oral administration there was significant improvement (p&lt;0.001) in the HDL‐inflammatory index (HII) in ApoE null mice. Oral L‐4F alone or niclosamide alone were ineffective. Subcutaneous injection of L‐4F alone but not niclosamide alone significantly improved the HII. Analysis of the L‐4F‐niclosamide complex by gradient gel electrophoresis or Fourier Transform Infrared Spectroscopy indicated that the self‐association of L‐4F was altered resulting in smaller micelles. Oral administration of L‐4F and niclosamide to old apoE null mice together with pravastatin resulted in regression of lesions. In another experiment ApoE null mice 3‐4 months of age were treated for 7 months with pravastatin in the drinking water and oral L‐4F together with niclosamide in doses which produced maximal L‐4F plasma concentrations of ~100 ng/mL. There was a significant reduction in lesion area compared to controls (p&lt;0.001). The reduction in lesion area was not improved by administering L‐4F subcutaneously for the first 3 months to achieve maximal L‐4F plasma concentrations ranging from 5 ‐ &gt;45 μg/mL. These results suggest that regimens achieving initial high L‐4F plasma concentrations followed by oral administration may be no more effective than oral treatment alone which produces maximal L‐4F plasma concentration.

IL-22 is induced by S100/calgranulin and impairs cholesterol efflux in macrophages by downregulating ABCG1

S100A8/9 and S100A12 are emerging biomarkers for disease activity of autoimmune and cardiovascular diseases. We demonstrated previously that S100A12 accelerates atherosclerosis accompanied by large cholesterol deposits in atherosclerotic lesions of apoE-null mice. The objective of this study was to ascertain whether S100/calgranulin influences cholesterol homeostasis in macrophages. Peritoneal macrophages from transgenic mice expressing human S100A8/9 and S100A12 in myeloid cells [human bacterial artificial chromosome (hBAC)/S100] have increased lipid content and reduced ABCG1 expression and [(3)H]cholesterol efflux compared with WT littermates. This was associated with a 6-fold increase in plasma interleukin (IL)-22 and increased IL-22 mRNA in splenic T cells. These findings are mediated by the receptor for advanced glycation endproducts (RAGE), because hBAC/S100 mice lacking RAGE had normal IL-22 expression and normal cholesterol efflux. In vitro, recombinant IL-22 reduced ABCG1 expression and [(3)H]cholesterol efflux in THP-1 macrophages, while recombinant S100A12 had no effect on ABCG1 expression. In conclusion, S100/calgranulin has no direct effect on cholesterol efflux in macrophages, but rather promotes the secretion of IL-22, which then directly reduces cholesterol efflux in macrophages by decreasing the expression of ABCG1.

Augmented atherogenesis in ApoE-null mice co-exposed to polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) are persistent organic pollutants found as complex mixtures in the environment throughout the world. Therefore, humans are ubiquitously and simultaneously exposed to TCDD and PCBs. TCDD and PCBs alone have been linked to atherosclerosis. However, the effects of interactions or synergism between TCDD and PCBs on atherogenesis are unknown. We investigated the possible enhanced atherogenesis by co-exposure to TCDD and PCBs and the potential mechanism(s) involved in this enhancement. Male ApoE−/− mice were exposed to TCDD (15μg/kg) and Aroclor1254 (55mg/kg, a representative mixture of PCBs) alone or in combination by intraperitoneal injection four times over six weeks of duration. Our results showed that mice exposed to TCDD alone, but not Aroclor1254 alone, developed atherosclerotic lesions. Moreover, we found that atherosclerotic disease was exacerbated to the greatest extent in mice co-exposed to TCDD and Aroclor1254. The enhanced lesions correlated with several pro-atherogenic changes, including a marked increase in the accumulation of the platelet-derived chemokine PF4, and the expression of the proinflammatory cytokine MCP-1 and the critical immunity gene-RIG-I. Our data demonstrated that co-exposure to TCDD and Aroclor1254 markedly enhanced atherogenesis in ApoE−/− mice. Significantly, our observations suggest that combined exposure to TCDD and PCBs may be a greater cardiovascular health risk than previously anticipated from individual studies.

Recoupling of eNOS with Folic Acid Prevents Abdominal Aortic Aneurysm Formation in Angiotensin II-Infused Apolipoprotein E Null Mice

We have previously shown that eNOS uncoupling mediates abdominal aortic aneurysm (AAA) formation in hph-1 mice. In the present study we examined whether recoupling of eNOS prevents AAA formation in a well-established model of Angiotensin II-infused apolipoprotein E (apoE) null mice by targeting some common pathologies of AAA. Infusion of Ang II resulted in a 92% incidence rate of AAA in the apoE null animals. In a separate group, animals were treated orally with folic acid (FA), which is known to recouple eNOS through augmentation of dihydrofolate reductase (DHFR) function. This resulted in a reduction of AAA rate to 19.5%. Imaging with ultrasound showed that FA markedly inhibited expansion of abdominal aorta. FA also abolished elastin breakdown and macrophage infiltration in the AAA animals. The eNOS uncoupling activity, assessed by L-NAME-sensitive superoxide production, was minimal at baseline but greatly exaggerated with Ang II infusion, which was completely attenuated by FA. This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability. Furthermore, the expression and activity of DHFR was decreased in Ang II-infused apoE null mice specifically in the endothelial cells, while FA administration resulted in its recovery. Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.

Endothelial nitric oxide synthase activation through obacunone-dependent arginase inhibition restored impaired endothelial function in ApoE-null mice

Endothelial arginase constrains the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion and reduces nitric oxide bioavailability. During the screening course of arginase inhibitor, we found obacunone as an arginase inhibitor. We tested the hypothesis that obacunone regulates vascular endothelial NO production. Obacunone incubation inhibited arginase I and II activities in liver and kidney lysates, respectively, in dose-dependent manner. Obacunone reciprocally increased nitrite/nitrate (NOx) production in HUVECs. In isolated aortic rings, obacunone increased intracellular l-arginine concentration and enhanced eNOS coupling, leading to increased NO and decreased superoxide production, with no changes in protein expression. Vasoconstriction response to U46619 was attenuated in obacunone-treated aortic vessels compared to that in untreated vessels. Endothelium-dependent vasorelaxant response to acetylcholine was significantly increased in obacunone-treated vessels and was modulated by the NO-dependent signaling cascade. The dose-dependent vasorelaxant response to Ach was reduced in the aortic vessels of ApoE−/− mice fed a high-cholesterol diet. Obacunone incubation increased vasorelaxation to the level of a WT mouse, although the endothelium-independent response to sodium nitroprusside was identical among the groups. Therefore, obacunone may help treat cardiovascular diseases derived from endothelial dysfunction and may be useful for designing pharmaceutical compounds.

The Proatherogenic Effect of Chronic Nitric Oxide Synthesis Inhibition in ApoE-Null Mice Is Dependent on the Presence of PPARα

Inhibition of endothelial nitric oxide synthase (eNOS) accelerates atherosclerosis in ApoE-null mice by impairing the balance between angiotensin II (AII) and NO. Our previous data suggested a role for PPARα in the deleterious effect of the renin-angiotensin system (RAS). We tested the hypothesis that ApoE-null mice lacking PPARα (DKO mice) would be resistant to the proatherogenic effect of NOS inhibition. DKO mice fed a Western diet were immune to the 23% worsening in aortic sinus plaque area seen in the ApoE-null animals under 12 weeks of NOS inhibition with a subpressor dose of L-NAME, P = 0.002. This was accompanied by a doubling of reactive oxygen species (ROS-) generating aortic NADPH oxidase activity (a target of AII, which paralleled Nox1 expression) and by a 10-fold excess of the proatherogenic iNOS, P < 0.01. L-NAME also caused a doubling of aortic renin and angiotensinogen mRNA level in the ApoE-null mice but not in the DKO, and it upregulated eNOS in the DKO mice only. These data suggest that, in the ApoE-null mouse, PPARα contributes to the proatherogenic effect of unopposed RAS/AII action induced by L-NAME, an effect which is associated with Nox1 and iNOS induction, and is independent of blood pressure and serum lipids.

Immunization with advanced glycation end products modified low density lipoprotein inhibits atherosclerosis progression in diabetic apoE and LDLR null mice

BackgroundDiabetes accelerates atherosclerosis through undefined molecular mechanisms. Hyperglycemia induces formation of advanced glycation end product (AGE)-modified low-density lipoprotein (LDL). Anti-AGE-LDL autoantibodies favor atherosclerosis (AS) progression in humans, while anti oxidized LDL immunization inhibits AS in hypercholesterolemic, non-diabetic mice. We here investigated if AGE-LDL immunization protects against AS in diabetic mice.MethodsAfter diabetes induction with streptozotocin and high fat diet, both low density lipoprotein receptor (LDLR)-/- and apoE female mice were randomized to: AGE-LDL immunization with aluminum hydroxide (Alum) adjuvant; Alum alone; or PBS.ResultsAGE-LDL immunization: significantly reduced AS; induced specific plasma IgM and IgG antibodies; upregulated splenic Th2, Treg and IL-10 levels, without altering Th1 or Th17 cells; and increased serum high density lipoprotein(HDL) while numerically lowering HbA1c levels.ConclusionsSubcutaneous immunization with AGE-LDL significantly inhibits atherosclerosis progression in hyperlipidemic diabetic mice possibly through activation of specific humoral and cell mediated immune responses and metabolic control improvement.

Abstract 18108: Decreased Fmo3 Expression is Associated With Increased Atherosclerosis and Impaired Liver Function in Apolipoprotein E Null Mice

Recent studies demonstrated a strong positive association between blood trimethylamine-N-oxide (TMAO) level and risk for cardiovascular disease. Dietary choline is converted to trimethylamine (TMA) by the gut bacteria, and TMA is then converted to TMAO in the liver, mainly by flavin containing monooxygenase 3 (Fmo3). To examine how FMO3 expression influences atherogenesis, we administered two different FMO3 or control antisense oligonucleotdies (ASOs) to apolipoprotein E (apoE) null mice for 15 weeks. During the last 12 weeks of the ASO treatment, the mice received a chow diet containing 1% choline to enhance TMA production. Hepatic FMO3 mRNA levels were decreased by 80% in mice that received either FMO3 ASO #1 or #2 versus those that received the control ASO. As expected, circulating TMA and TMAO levels were significantly increased and decreased, respectively, in the mice that received the two FMO3 ASOs versus the controls. Other observed changes in the FMO3 ASO-treated mice included: decreased plasma triglyceride, HDL, and glucose levels. Significantly increased VLDL/IDL/LDL cholesterol was observed only in FMO3 ASO #2-treated mice versus the controls. FMO3 ASO treatment increased circulating total bile acid levels by more than 2-fold. Compared to controls, serum markers of hepatic inflammation and necrosis (ALT, AST) were significantly increased in FMO3 ASO treated mice. Histological examination revealed obvious hepatic steatosis and increased inflammatory cell infiltration, suggesting that FMO3 abrogation may be a reasonable model for non-alcoholic steatohepatitis (NASH). We also found a significant increase in quantitative triglyceride and cholesterol content of these livers. Transcriptional profiling by microarrays showed increased hepatic expression of inflammatory genes in mice that received FMO3 ASOs versus the controls. In addition, a significant increase in spleen weight in these mice suggests increased systemic inflammation. Finally, FMO3 knockdown increased atherosclerosis by more than 50% when compared to controls (p &lt; 0.01). This work demonstrates that blocking FMO3 activity has a major impact on liver physiology and atherosclerosis. Our study suggests a protective role for FMO3 in atherosclerosis and liver function.

PKCβ Promotes Vascular Inflammation and Acceleration of Atherosclerosis in Diabetic ApoE Null Mice

Subjects with diabetes mellitus are at high risk for developing atherosclerosis through a variety of mechanisms. Because the metabolism of glucose results in production of activators of protein kinase C (PKC)β, it was logical to investigate the role of PKCβ in modulation of atherosclerosis in diabetes mellitus. ApoE(-/-) and PKCβ(-/-)/ApoE(-/-) mice were rendered diabetic with streptozotocin. Quantification of atherosclerosis, gene expression profiling, or analysis of signaling molecules was performed on aortic sinus or aortas from diabetic mice. Diabetes mellitus-accelerated atherosclerosis increased the level of phosphorylated extracellular signal-regulated kinase 1/2 and Jun-N-terminus kinase mitogen-activated protein kinases and augmented vascular expression of inflammatory mediators, as well as increased monocyte/macrophage infiltration and CD11c(+) cells accumulation in diabetic ApoE(-/-) mice, processes that were diminished in diabetic PKCβ(-/-)/ApoE(-/-) mice. In addition, pharmacological inhibition of PKCβ reduced atherosclerotic lesion size in diabetic ApoE(-/-) mice. In vitro, the inhibitors of PKCβ and extracellular signal-regulated kinase 1/2, as well as small interfering RNA to Egr-1, significantly decreased high-glucose-induced expression of CD11c (integrin, alpha X 9 complement component 3 receptor 4 subunit]), chemokine (C-C motif) ligand 2, and interleukin-1β in U937 macrophages. These data link enhanced activation of PKCβ to accelerated diabetic atherosclerosis via a mechanism that includes modulation of gene transcription and signal transduction in the vascular wall, processes that contribute to acceleration of vascular inflammation and atherosclerosis in diabetes mellitus. Our results uncover a novel role for PKCβ in modulating CD11c expression and inflammatory response of macrophages in the development of diabetic atherosclerosis. These findings support PKCβ activation as a potential therapeutic target for prevention and treatment of diabetic atherosclerosis.

Abstract 367: Macrophage 12/15-lipoxygenase Mediates Insulin-like Growth Factor I (IGF-1)-induced Suppression of Foam Cells: Potential Mechanism Responsible for IGF-1-induced Atheroprotection

We have shown that IGF-1 infusion into Apoe-null mice suppressed atherosclerosis. To define mechanisms we performed RT-PCR array and found that IGF-1 (0.8 mg/kg, i.p, 7d) reduced aortic 12/15-lipoxygenase (LOX) expression (64±6% decrease vs. saline control, P&lt;0.05). Macrophage LOX is a major pro-atherogenic enzyme that mediates oxidation of native low density lipoprotein (nLDL) and via this mechanism LOX promotes lipid uptake by macrophages (MΦ) and formation of foam cells. IGF-1 infusion into Apoe-null mice (1.5 mg/kg/d, 12 wks on a Western diet) reduced LOX plaque immunopositivity (74±9% decrease, P&lt;0.05), suppressed plaque oxidative stress (64±7% decrease, IHC with 8-oxodG a/b, P&lt;0.05), reduced plaque lipids (35%±6 decrease, Oil Red staining, P&lt;0.05) and decreased atherosclerotic lesion area in aortic valve (IGF-1: 0.259 ± 0.018 mm2, saline: 0.356 ± 0.031 mm2; P&lt;0.05). To further define mechanisms THP-1-derived MΦ were exposed to IGF-1. IGF-1 (50 ng/ml, 16h) reduced LOX mRNA (49±6% decrease, RT-PCR, P&lt;0.05), decreased LOX protein (42±6% decrease, immunoblotting with LOX a/b, P&lt;0.05) and reduced LOX activity (32±5% decrease, 15S-HETE ELISA, P&lt;0.05) and IGF-1 suppressed STAT6 transcription factor binding to LOX gene promoter region (CHIP assay with STAT6 a/b). IGF-1 markedly reduced oxidation of nLDL mediated by THP-1 or mouse peritoneal MΦ (85±6% and 67±5% decrease, respectively, MDA assay). IGF-1 decreased subsequent LDL uptake (i.e. formation of foam cells) mediated by THP-1 or WT peritoneal MΦ (45±8% and 51±3% decrease, respectively, Oil Red staining); however both lipid uptake and IGF-1 effect were decreased in peritoneal MΦ isolated from LOX-null mice (P&gt;0.05). These data indicate that specific LOX downregulation mediates IGF-1-induced suppression of nLDL oxidation and formation of foam cells. In summary, IGF-1 reduced LOX expression in atherosclerotic plaque and this effect correlated with reduction in plaque lipids and in total atherosclerotic burden. IGF-1 downregulated LOX in cultured MΦ via inhibition of STAT6-dependent transcription and IGF-1 decreased lipid oxidation and uptake by MΦ. Taken together these data strongly suggest that MΦ LOX downregulation mediates IGF-1-induced reduction in foam cells and in atherosclerosis.

Abstract 138: Treatment of Atherosclerosis by Systemic Protease Inhibition

Background Elevated activity of urokinase plasminogen activator (uPA) and matrix metalloproteases (MMPs) in human arteries is associated with accelerated atherosclerosis (athero), aneurysms, and plaque rupture. Apoe -null mice with macrophage-targeted uPA overexpression (SR-uPA mice) have elevated vascular uPA and MMP activity, accelerated athero, dilated aortic roots, and increased histologic features of plaque rupture. SR-uPA mice are therefore a useful experimental setting for beginning to investigate whether inhibition of uPA or MMPs can prevent athero progression, aortic dilation, and plaque rupture. Hypothesis Treatment with a murine monoclonal antibody inhibiting mouse uPA (mU1), or a limited-spectrum synthetic MMP inhibitor (XL784) will reduce athero in SR-uPA mice. Methods SR-uPA mice were fed a high-fat diet for 10 wk beginning at 5 - 6 wk of age. Fat-fed mice were injected with mU1 or control antibody twice per wk (for 10 wk), or were given either XL784 (125, 250, or 500 mg/kg) or vehicle by daily oral gavage. Mice were then anesthetized, formalin-perfused, and athero was measured in isolated aortic roots and pinned aortas. Groups were compared by t-test and ANOVA. Results None of the treatments altered peripheral blood monocyte counts or plasma cholesterol. mU1 reduced aortic root intimal lesion area by 20% ( P &lt; 0.05) and aortic root circumference by 12% ( P = 0.01). All 3 doses of XL784 significantly decreased oil-red O-positive lesion area (36 - 42%; P &lt; 0.05 for all). All XL784 doses also reduced aortic root intimal lesion area (22 - 29%; P &lt; 0.05 for low dose only). The highest XL784 dose also reduced total macrophage-positive lesion area (33%; P &lt; 0.05). All XL784 doses yielded trends towards decreased aortic root circumference (5 - 10%). Neither mU1 nor XL784 significantly altered percent aortic surface lesion coverage. Conclusions In SR-uPA mice, pharmacologic inhibition of either uPA or selected MMPs significantly decreased aortic root athero and limited aortic dilation. Differential effects of both agents on aortic root versus surface athero suggest prevention of athero progression rather than initiation. These approaches to protease inhibition show promise for preventing the progression and complications of established athero.

Abstract 470: Aip1 Suppresses Atherosclerosis by Limiting Hyperlipidemia-Induced Inflammation and Vascular Endothelial Dysfunction

Objective- Apoptosis signal-regulating kinase 1 (ASK1)-interacting protein-1 (AIP1) is a signaling adaptor molecule implicated in stress and apoptotic signaling induced by proinflammatory mediators. However, its function in atherosclerosis has not been established. In the present study, we use AIP1-null (AIP1-/-) mice to examine its effect on atherosclerotic lesions in an ApoE-null (ApoE-/-) mouse model of atherosclerosis. Methods and Results- ApoE-/- control mice developed atherosclerosis in the aortic roots and descending aortas upon Western-type diet for 10 weeks, while the atherosclerotic lesions are significantly augmented in ApoE-/-AIP1-/- double knockout (DKO) mice. DKO mice show increases in plasma inflammatory cytokines with no significant alterations in body weight, total cholesterol levels or lipoprotein profiles. Aortas in DKO mice show increased inflammation and endothelial cell (EC) dysfunction with NF-κB activity, correlating with increased accumulation of macrophages in the lesion area. Importantly, macrophages from DKO donors are not sufficient to augment inflammatory responses and atherogenesis when transferred to ApoE-KO recipients. Mechanistic studies suggest that AIP1 is highly expressed in aortic EC but not in macrophages, and AIP1 deletion in EC significantly enhance oxidized LDL-induced NF-κB signaling, gene expression of inflammatory molecules and monocyte adhesion, suggesting that vascular EC are responsible for the increased inflammatory responses observed in DKO mice. Conclusion- Our data demonstrate that loss of AIP1 in aortic EC primarily contributes to the exacerbated lesion expansion in the ApoE-/-AIP1-/- mice, revealing an important role of AIP1 in limiting inflammation, EC dysfunction and atherosclerosis.

Induction of C-X-C Chemokine Receptor Type 7 (CXCR7) Switches Stromal Cell-derived Factor-1 (SDF-1) Signaling and Phagocytic Activity in Macrophages Linked to Atherosclerosis

The discovery of CXCR7 as a new receptor for SDF-1 places many previously described SDF-1 functions attributed to CXCR4 in question, though whether CXCR7 acts as a signaling or "decoy" receptor has been in debate. It is known that CXCR7 is not expressed in normal blood leukocytes; however, the potential role of leukocyte CXCR7 in disease states has not been addressed. The aim of this study was to determine the expression and function of macrophage CXCR7 linked to atherosclerosis. Here, we show that CXCR7 was detected in macrophage-positive area of aortic atheroma of ApoE-null mice, but not in healthy aorta. During monocyte differentiation to macrophages, CXCR7 was up-regulated at mRNA and protein levels, with more expression in M1 than in M2 phenotype. In addition, CXCR7 induction was associated with a SDF-1 signaling switch from the pro-survival ERK and AKT pathways in monocytes to the pro-inflammatory JNK and p38 pathways in macrophages. The latter effect was mimicked by a CXCR7-selective agonist TC14012 and abolished by siRNA knockdown of CXCR7. Furthermore, CXCR7 activation increased macrophage phagocytic activity, which was suppressed by CXCR7 siRNA silencing or by inhibiting either the JNK or p38 pathways, but was not affected by blocking CXCR4. Finally, activation of CXCR7 by I-TAC showed a similar signaling and phagocytic activity in macrophages with no detectable CXCR3. We conclude that CXCR7 is induced during monocyte-to-macrophage differentiation, which is required for SDF-1 and I-TAC signaling to JNK and p38 pathways, leading to enhanced macrophage phagocytosis, thus possibly contributing to atherogenesis.

Type VIII Collagen Mediates Vessel Wall Remodeling after Arterial Injury and Fibrous Cap Formation in Atherosclerosis

Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions invivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.

Apolipoprotein E deficiency and a mouse model of accelerated liver aging

The liver is the central metabolic organ which regulates several key aspects of lipid metabolism. The liver changes with age leading to an impaired ability to respond to hepatic insults and an increased incidence of liver disease in the elderly. Apolipoprotein E (ApoE) null mice have proved to be a very popular model to study spontaneous atherosclerosis, but recently it has been demonstrated that in ApoE-/- mice liver there are enzymatic and structural alterations, normally linked to the age. The purpose of this study was to consider ApoE-/- mice as a model for oxidative stress induced hepatic disease and to clarify how ApoE inactivation accelerates the aging process and causes liver disease.We used ApoE null mice and control mice at different ages (6weeks and 15months).Liver morphological damage as well as proteins involved in oxidative stress and liver ageing were all analyzed.Our study showed that ApoE null mice develop important age-related changes including oxidative stress, pseudocapillarization, increased polyploidy, decreased hepatocyte number and increased nuclear size. Our findings provide evidence that hypercholesterolemic ApoE-/- mice are more likely to develop severe liver injury, suggesting that in addition to vascular disease, increased cholesterol products and oxidative stress may also play a role in accelerating the progression of aging in the liver.

Influence of apolipoprotein A-V on the metabolic fate of triacylglycerol

Apolipoprotein (apo) A-V functions to modulate intracellular and extracellular triacylglycerol metabolism. The present review addresses molecular mechanisms underlying these effects. The relevance of apoA-V to human disease conditions is illustrated by the strong correlation between single nucleotide polymorphisms in APOA5, elevated plasma triacylglycerol and dyslipidemic disease. Despite undergoing processing for secretion from hepatocytes, a portion of apoA-V escapes this destiny and accumulates as a component of cytosolic lipid droplets. Expression of recombinant apoA-V in hepatocarcinoma cells results in increased lipid droplet size and number at the expense of triacylglycerol secretion.ApoA-V modulates atherosclerosis in hypercholesterolemic apoE null mice. ApoE null/human apoA-V transgenic mice had reduced levels of triacylglycerol and cholesterol in plasma along with decreased aortic lesion size. ApoA-V modulates triacylglycerol metabolic fate. Following its synthesis, apoA-V enters the endoplasmic reticulum and associates with membrane defects created by triacylglycerol accumulation. Association of apoA-V with endoplasmic reticulum membrane defects promotes nascent lipid droplets budding toward the cytosol. Despite its low concentration in plasma (∼150 ng/ml), apoA-V modulates lipoprotein metabolism by binding to glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1. This interaction effectively localizes triacylglycerol-rich lipoproteins in the vicinity of glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein1's other ligand, lipoprotein lipase.