Lipoprotein Receptor-deficient Mice Research Articles

Mitochondrial apolipoprotein A-I binding protein alleviates atherosclerosis by regulating mitophagy and macrophage polarization

Apolipoprotein A-I binding protein (AIBP), a secreted protein, has been shown to play a pivotal role in the development of atherosclerosis. The function of intracellular AIBP, however, is not yet well characterized. Here, we found that AIBP is abundantly expressed within human and mouse atherosclerotic lesions and exhibits a distinct localization in the inner membrane of mitochondria in macrophages. Bone marrow-specific AIBP deficiency promotes the progression of atherosclerosis and increases macrophage infiltration and inflammation in low-density lipoprotein receptor-deficient (LDLR−/−) mice. Specifically, the lack of mitochondrial AIBP leads to mitochondrial metabolic disorders, thereby reducing the formation of mitophagy by promoting the cleavage of PTEN-induced putative kinase 1 (PINK1). With the reduction in mitochondrial autophagy, macrophages polarize to the M1 proinflammatory phenotype, which further promotes the development of atherosclerosis. Based on these results, mitochondrial AIBP in macrophages performs an antiatherosclerotic role by regulating of PINK1-dependent mitophagy and M1/M2 polarization.9wPYiwBcJkigAW4BX-B6DEVideo

Clinical Significance of HSCARG for Atherosclerotic Coronary Heart Disease and Reduced ROS-Oxidative Stress in in Vivo and in Vitro Models via p47phox by NF-κB Activity.

Coronary heart disease (CHD) is a dynamic process in which there are interactions between endothelial dysfunction, oxidative stress, and inflammatory responses. The aim of the present study was to investigate the function and mechanism of HSCARG in the treatment of CHD. Male apolipoprotein E/low-density lipoprotein receptor-deficient mice were given a high-fat diet with 21% fat and 0.15% cholesterol for the in vivo model. Human umbilical vein endothelial cells were incubated with angiotensin II for the in vitro model. HSCARG expression was inhibited in patients or mice with CHD. HSCARG reduced oxidative stress in mice with CHD. HSCARG also reduced reactive oxygen species (ROS)-oxidative stress in the in vitro model. HSCARG induced p47phox expression in the in vitro model by NF-κB activity. The regulation of nuclear factor kappa B (NF-κB) activity or p47phox expression participates in the effects of HSCARG in CHD. Altogether, our data indicate that HSCARG reduced ROS-oxidative stress in in vivo and in vitro models of CHD via p47phox by NF-κB activity and may be a clinical target for CHD.

(Pro)renin Receptor Inhibition Reduces Plasma Cholesterol and Triglycerides but Does Not Attenuate Atherosclerosis in Atherosclerotic Mice.

Objective: Elevated plasma cholesterol concentrations contributes to ischemic cardiovascular diseases. Recently, we showed that inhibiting hepatic (pro)renin receptor [(P)RR] attenuated diet-induced hypercholesterolemia and hypertriglyceridemia in low-density lipoprotein receptor (LDLR) deficient mice. The purpose of this study was to determine whether inhibiting hepatic (P)RR could attenuate atherosclerosis.Approach and Results: Eight-week-old male LDLR−/− mice were injected with either saline or N-acetylgalactosamine-modified antisense oligonucleotides (G-ASOs) primarily targeting hepatic (P)RR and were fed a western-type diet (WTD) for 16 weeks. (P)RR G-ASOs markedly reduced plasma cholesterol concentrations from 2,211 ± 146 to 1,128 ± 121 mg/dL. Fast protein liquid chromatography (FPLC) analyses revealed that cholesterol in very low-density lipoprotein (VLDL) and intermediate density lipoprotein (IDL)/LDL fraction were potently reduced by (P)RR G-ASOs. Moreover, (P)RR G-ASOs reduced plasma triglyceride concentrations by more than 80%. Strikingly, despite marked reduction in plasma lipid concentrations, atherosclerosis was not reduced but rather increased in these mice. Further testing in ApoE−/− mice confirmed that (P)RR G-ASOs reduced plasma lipid concentrations but not atherosclerosis. Transcriptomic analysis of the aortas revealed that (P)RR G-ASOs induced the expression of the genes involved in immune responses and inflammation. Further investigation revealed that (P)RR G-ASOs also inhibited (P)RR in macrophages and in enhanced inflammatory responses to exogenous stimuli. Moreover, deleting the (P)RR in macrophages resulted in accelerated atherosclerosis in WTD fed ApoE−/− mice.Conclusion: (P)RR G-ASOs reduced the plasma lipids in atherosclerotic mice due to hepatic (P)RR deficiency. However, augmented pro-inflammatory responses in macrophages due to (P)RR downregulation counteracted the beneficial effects of lowered plasma lipid concentrations on atherosclerosis. Our study demonstrated that hepatic (P)RR and macrophage (P)RR played a counteracting role in atherosclerosis.

Intermittent Fasting Inhibits High-Fat Diet-Induced Atherosclerosis by Ameliorating Hypercholesterolemia and Reducing Monocyte Chemoattraction.

Atherosclerosis is a major pathology for cardiovascular diseases (CVDs). Clinically, the intermittent fasting (IF) has been observed to reduce the risk of CVDs. However, the effect of IF on the development of atherosclerosis has not been fully elucidated. Herein, we determined the protection of IF against high-fat diet–induced atherosclerosis in pro-atherogenic low-density lipoprotein receptor deficient (LDLR-/-) mice and the potentially involved mechanisms. The LDLR-/- mice were scheduled intermittent fasting cycles of 3-day HFD feeding ad libitum and 1 day fasting, while the mice in the control group were continuously fed HFD. The treatment was lasted for 7 weeks (∼12 cycles) or 14 weeks (∼24 cycles). Associated with the reduced total HFD intake, IF substantially reduced lesions in the en face aorta and aortic root sinus. It also increased plaque stability by increasing the smooth muscle cell (SMC)/collagen content and fibrotic cap thickness while reducing macrophage accumulation and necrotic core areas. Mechanistically, IF reduced serum total and LDL cholesterol levels by inhibiting cholesterol synthesis in the liver. Meanwhile, HFD-induced hepatic lipid accumulation was attenuated by IF. Interestingly, circulating Ly6Chigh monocytes but not T cells and serum c-c motif chemokine ligand 2 levels were significantly reduced by IF. Functionally, adhesion of monocytes to the aortic endothelium was decreased by IF via inhibiting VCAM-1 and ICAM-1 expression. Taken together, our study indicates that IF reduces atherosclerosis in LDLR-/- mice by reducing monocyte chemoattraction/adhesion and ameliorating hypercholesterolemia and suggests its potential application for atherosclerosis treatment.

Abstract MP62: Sars-cov-2 Spike Protein Regulation Of Angiotensin Converting Enzyme 2 And Tissue Renin-angiotensin Systems: Influence Of Biologic Sex

Angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor and an enzyme of the renin-angiotensin system (RAS), is on the X chromosome and stimulated by estrogen. Male sex is a risk factor for SARS-CoV-2 severity. Previous investigators demonstrated that the SARs-CoV-2 Spike (S) protein decreases tissue ACE2 by protein internalization or shedding. This study defined sex differences in tissue ACE2 expression and their impact on SARS-CoV-2 S protein regulation of ACE2 activity and AngII levels. Male and female intact or gonadectomized (GDX) low density lipoprotein receptor deficient ( Ldlr -/- ) mice, and Four Core Genotype (FCG) male (XY or XX) or female (XX or XY) mice were fed a Western diet for 4 months. In lung, ACE2 mRNA abundance was similar in male and female mice and reduced by GDX (Male XY intact: 1.04 ± 0.15; Female XX intact: 1.13 ± 0.13; Male XY GDX: 0.11 ± 0.03; Female XX GDX: 0.18 ± 0.04 ΔΔCt; P<0.05). Lungs from XX mice had higher ACE2 mRNA abundance than XY mice regardless of gonadal sex (P<0.05), and GDX reduced ACE2 mRNA abundance in lungs of XX, but not XY females (XX Female GDX: 0.18 ± 0.04; XY Female GDX: 0.38 ± 0.09; P<0.05). In adipose, XX females had higher ACE2 mRNA abundance than XY males (XX female: 5.4 ± 0.7; XY male: 1.0 ± 0.1; P<0.05), regardless of gonadal sex (XY females: 3.3 ± 0.7; XX males: 1.5 ± 0.3; P<0.05). Male XY and female XX Ldlr -/- mice were administered vehicle or SARS-CoV-2 S protein (2 nmol/kg, ip, 3 doses) with tissue harvest six hours later. In lung, AngII levels were increased by S protein in male, but not female mice (Male, vehicle: 12.3 ± 2.3; Male, S protein: 33.6 ± 7.1; Female, vehicle: 16.1 ± 2.0; Female, S protein: 20.2 ± 1.3 pg/μg protein; P<0.05). In adipose, ACE2 activity was reduced by S protein in male, but not female mice (Male, vehicle: 63.6 ± 13.9; Male, S protein: 26.1 ± 1.9; Female, vehicle: 32.5 ± 1.9; Female, S protein: 25.1 ± 1.3 RFU/hr/mg tissue; P<0.05). SARS-CoV-2 S protein (35 nM) decreased ACE2 activity in type II lung alveolar cells (Vehicle: 2.0 x 10 4 ; S protein: 1.2 x 10 4 RFU/10 6 cells) and 3T3-L1 adipocytes (Vehicle: 2.1 x 10 4 ± 0.3 x 10 4 ; S protein: 1.1 x 10 4 ± 0.8 x 10 3 RFU/10 5 cells; P<0.05). Biologic sex regulation of ACE2 may protect females from SARS-CoV-2 S protein-mediated ACE2 reductions and activation of the local RAS.

Chronic radiation exposure aggravates atherosclerosis by stimulating neutrophil infiltration

Background Radiation exposure is known to increase the risk of chronic inflammatory diseases, such as atherosclerosis, by modulating inflammation. Methods To investigate the infiltration of leukocytes in radiation-aggravated atherosclerosis, we examined low-density lipoprotein receptor-deficient (Ldlr–/–) mice and C57BL/6j mice after exposure to 0.5 or 1 Gy radiation over 16 weeks. Results We found that radiation exposure induced atherosclerosis development in Ldlr–/– mice, as demonstrated by increased lipid-laden plaque size, reactive oxygen species levels, and levels of the pro-inflammatory cytokines, IL-1β and TNF-α, in the aortas and spleens. Total plasma cholesterol, triglyceride, and LDL cholesterol levels were also increased by radiation exposure, along with cardiovascular risk. We also showed dose-dependent increases in neutrophils and monocytes that coincided with a reduction in lymphocytes in the spleens of Ldlr–/– mice. The correlation between the infiltration of leukocytes and cytokine production was also confirmed in the hearts and spleens of these mice. Conclusions We concluded that chronic radiation exposure increased the production of pro-inflammatory mediators, which was associated with the migration of neutrophils and inflammatory monocytes into sites of atherosclerosis. Thus, our data suggest that the accumulation of neutrophils and inflammatory monocytes, together with the reduction of lymphocytes, contribute to aggravated atherosclerosis in Ldlr–/– mice under prolonged exposure to radiation.

B cell-specific GPR55 deficiency promotes atherosclerosis

Abstract Background Atherosclerosis is accompanied by an imbalance between resolving and pro-inflammatory lipid mediators. Targeting lipid signaling pathways might offer a new anti-inflammatory therapy for improving the clinical outcome in cardiovascular disease patients. We considered lysophosphatidylinositol (LPI) and its receptor G protein-coupled receptor (GPR)55 as a potential modulator of atherosclerosis. Its role in regulating atherosclerosis and B cell function is unknown. Hypothesis We assessed the hypothesis that GPR55 signaling causally affects atherosclerosis and whether it has a specific role in regulating B cell function in this disease. Methods Atherosclerotic plaques were compared between apolipoprotein E deficient (ApoE−/−) and ApoE−/−Gpr55−/− mice after 4 to 16 weeks Western Diet (WD; 0.15% cholesterol; n=12–15 per group). To specifically test the role of B cell GPR55 in atherosclerosis, we generated mixed chimeras by lethally irradiating low density lipoprotein receptor deficient (Ldlr−/−) mice and reconstituting with a mixture of μMT and wildtype (control) or μMT and Gpr55−/− bone marrow cells. Circulating B cells were sorted and bulk RNA sequencing analysis was performed. We performed lipid and immunostainings of murine aortic root plaques, qPCR and ELISA of tissue lysates, as well as multiplex analysis of plasma immunoglobulins. Leukocyte plasma and tissue counts were determined by flow cytometry. Results GPR55 expression in mouse and human atherosclerotic plaques was detected by immunostaining. Furthermore, we confirmed murine Gpr55 mRNA expression on sorted circulating B220+B cells via qPCR, which was higher compared to CD3+ T cells, while CD11+ myeloid cells as well as NK cells had only low Gpr55 mRNA levels. ApoE−/−Gpr55−/− mice had significantly larger plaques after 4&16 weeks WD compared to ApoE−/− controls, with more pronounced body weight increases and higher cholesterol levels at the 16 weeks WD time point. In addition, global Gpr55 deficiency resulted in enhanced aortic pro-inflammatory cytokine mRNA expression (IL-1β, IL-6, TNFα) and a massively upregulated IgG1 plasma levels and increased percentages of splenic germinal center and plasma cells. B-cell RNA-seq analysis showed 460 differential expressed regulated genes in the ApoE−/−Gpr55−/− compared to ApoE−/−. The main pathways affected were calcium ion transport, immunoglobulin production, negative regulation of phosphorylation, and cellular component morphogenesis, suggesting a dsysregulation of B cell function. B cell specific Gpr55 deficiency blunted the metabolic effects on body weight and cholesterol, but still translated in larger atherosclerotic plaques and elevated plasma IgG levels compared to the respective controls. Conclusion Both global and B cell-restricted Gpr55 deficiency promotes atherosclerosis and is associated with a more pro-inflammatory phenotype. Our findings suggest a novel role for GPR55 in regulating B cell development and function. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

P4141Lack of IkBNS promotes cholate-containing high-fat diet-induced inflammation and atherogenesis in low-density lipoprotein (LDL) receptor-deficient mice

Abstract Background IκBNS is one of the nuclear IκB proteins and regulates a subset of Toll-like receptor (TLR) dependent genes. LPS acts as extremely strong stimulator of innate immunity. We tried to investigate whether stimulation of innate immunity could promote atherosclerosis in the IκBNS-deficient atherogenic mice. However all IκBNS-deficient mice died of LPS challenge at a dose of which almost all wild-type mice survived, because IκBNS-deficient mice are highly sensitive to LPS-induced endotoxin shock. Then, we decided to use a cholate-containing high fat diet (HFD(CA(+))), which has been widely used as an atherogenic diet in mice. Furthermore, HFD(CA(+)) has been shown to induce TLR4 mediated early inflammatory response. The present study aims to clarify the lack of IκBNS promotes atherogenesis in LDL receptor-deficient (LDLr−/−) mice fed HFD(CA(+)) compared with those fed a cholate-free HFD (HFD(CA(−)). Methods and results Mice that lacked IκBNS (IκBNS−/−) were crossed with LDLr−/− mice and formation of atherosclerotic lesions was analyzed after 6 weeks consumption of HFD(CA(+)) or HFD(CA(−)). The extent of atherosclerosis in the aorta (en face) was significantly increased in IκBNS−/−/LDLr−/−(CA(+)) mice compared with others after 6-week consumption of HFD (p<0.01) (Figure). Interestingly, HFD(CA(−)) did not induce significant atherosclerotic lesions in IκBNS−/−/LDLr−/− compared with LDLr−/− mice after 6-week consumption (Figure). Immunostaining of aortic root lesion revealed that HFD(CA(+)) significantly increased positive area of Mac-3 (macrophage) by 1.5-fold (p=0.01) and TLR4, interleukin-6 (IL-6) expression by 1.7-fold (P<0.05) and 1.5-fold (p<0.05) respectively in IκBNS−/−/LDLr−/− (CA(+)) compared to LDLr−/− (CA(+)) mice. Furthermore, active STAT3 (pSTAT3)-positive cells were significantly increased by 1.7-fold in the atherosclerotic lesions of IκBNS−/−/LDLr−/− (CA(+)) compared with LDLr−/− (CA(+)) mice (p<0.01). TLR4 positive areas, IL-6 positive areas, and pSTAT3 positive cells were overlapped with Mac-3, indicating that TLR4-IL-6-STAT3 axis was activated in macrophages in IκBNS−/−/LDLr−/− (CA(+)) mice. On the other hand, HFD(CA(−)) could not induce any difference in these immunoreactivities of arteriosclerotic lesions between IκBNS−/−/LDLr−/− (CA(−)) compared with LDLr−/− (CA(−)) mice. These findings suggest that IκBNS deficiency and HFD(CA(+)) promote atherogenesis in LDLr−/− mice via TLR4/IL-6/STAT3 pathway. Finally, we show the monocytes from peripheral blood of IκBNS−/−/LDLr−/− (CA(+)) mice were found to contain the most mounts of Ly6Chi among four groups, suggesting that lack of IκBNS enhances inflammation in the response HFD(CA(+)) feeding and thereby influence atherogenesis in IκBNS−/−/LDLr−/− mice. Aortic root atherosclerotic lesions Conclusions The present study is the first to demonstrate that the activation of innate immune system using HFD(CA(+)) induced significant inflammation and atherogenesis in IκBNS−/−/LDLr−/− compared with LDLr−/− mice.

Abstract 524: Fibrinogen Depletion Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm

Background: Fibrinogen and fibrin provide physical and biochemical support to a developing clot and is defined as one of the most crucial independent risk factors for cardiovascular diseases (CVDs). In addition to clot formation, fibrinogen promotes wound healing and powerful inflammatory and immune responses by engagement of leukocytes. Increased circulating fibrinogen and fibrin degradation products are correlated with increased diameter and progression of abdominal aortic aneurysm (AAA). However, a causal link between fibrinogen and AAA has not yet been established. The objective of this study was to determine the role of fibrinogen depletion in a mouse model of AAA. Methods and results: To determine whether aneurysm resulted in a procoagulant environment, we examined plasma levels of thrombin generation by calibrated automated thrombography (CAT), thrombin anti-thrombin (TAT), and fibrinogen in control and AAA plasma from mice and humans. Patients and mice with AAA had significant elevations in thrombin generation, TAT, and fibrinogen versus saline controls (mice) and control patients (human). To determine the effect of fibrinogen, in vivo, low density lipoprotein receptor deficient ( Ldlr -/- ) mice were injected with scrambled anti-sense oligonucleotide (ASO) or β-fibrinogen ASO (30 mg/kg) 3 weeks prior to experimentation and throughout the study. Fibrinogen ASO treatment achieved > 90% depletion of fibrinogen. After 3 weeks, mice were fed a fat and cholesterol enriched diet (42% milk fat; 0.2% cholesterol) 1 week prior to and throughout infusion with angiotensin II (AngII; 1,000 ng/kg/day) for 28 days. Fibrinogen ASO attenuated abdominal diameter (33% decrease; P = 0.001), and inflammatory cytokines (>75% decreased IL-1 and IL-6; P = 0.001) versus scrambled ASO control. Further, fibrinogen depletion significantly attenuated aneurysm incidence and rupture-induced death (P < 0.05). Conclusions: Our results demonstrate that AAAs augment procoagulant markers in both humans and mice. Importantly, fibrinogen depletion attenuates AAA incidence, diameter, rupture-induced death, and inflammation. Therefore, reduction of plasma fibrinogen may be a novel treatment strategy in patients with AAA.

Abstract 632: Myeloid beta catenin Deficiency Exacerbates Atherosclerosis in Low-density Lipoprotein-receptor Deficient Mice

Objective: The Wnt/β-catenin signaling is an ancient and evolutionarily conserved pathway that regulates essential aspects of cell differentiation, proliferation, migration and polarity, and canonical Wnt/β-catenin signaling has also been implicated in the pathogenesis of atherosclerosis. Macrophage is one of the major cell types involved in the initiation and progression of atherosclerosis, but the role of macrophage β-catenin in atherosclerosis remains elusive. This study aims to investigate the impact of β-catenin expression on macrophage functions and atherosclerosis development. Approaches and Results: To investigate the role of macrophage canonical Wnt/β-catenin signaling in atherogenesis, we generated low-density lipoprotein receptor-deficient (LDLR -/- ) mice with myeloid-specific β-catenin deficiency. As expected, deletion of β-catenin decreased macrophage adhesion and migration properties in vitro. However, deficiency of β-catenin significantly increased atherosclerosis in LDLR -/- mice without altering the plasma lipid levels. Mechanistic studies revealed that β-catenin can regulate activation of signal transducer and activator of transcription (STAT) pathway in macrophages, and ablation of β-catenin resulted in STAT3 downregulation and STAT1 activation, leading to elevated macrophage inflammatory responses and increased atherosclerosis. Conclusions: This study demonstrates a critical role of myeloid β-catenin expression in atherosclerosis by modulating macrophage inflammatory responses.

Abstract 422: Protease-Activated Receptor 2 is Critical for the Formation and Progression of Atherosclerosis

Objective: Protease-activated receptor 2 (PAR-2)-dependent signaling results in augmented inflammation and has been implicated in the pathogenesis of several autoimmune conditions. While PAR-2 protein is present in coronary atherosclerotic lesions, the relevance of this finding has not been investigated in experimental models. The objective of this study was to determine the effects of PAR-2 on the development of atherosclerosis. Methods and Results: Relative expression of PAR-2 is increased in human coronary artery (21 fold) and mouse aortic arch (16 fold) atheromas versus control coronary and aortic arch arteries, respectively (P = 0.001). To determine the effect of PAR-2 deficiency on atherosclerosis, male low density lipoprotein receptor deficient ( Ldlr -/- ) mice (8-12 weeks old) that were Par-2 +/+ or Par-2 -/- were fed a fat and cholesterol-enriched diet for 12 (n = 10 each group) or 24 weeks (n = 5 each group). PAR-2 deficiency attenuated atherosclerosis in the aortic sinus and aortic root with no effects on total plasma cholesterol concentrations or lipoprotein distributions after 12 (P = 0.000433) and 24 (P = 0.037) weeks. These reductions were attributable to both hematopoietic and non-hematopoietic-derived PAR-2 from analysis of bone marrow experiments (n = 15 for each of 4 chimeric groups; P < 0.05). Mechanistic studies using ex vivo macrophages show that activation of PAR-2 results in augmented foam cell formation and apoptosis with treatment of oxidized low-density lipoprotein in conjunction with decreased expression of the nuclear receptor LXR-alpha and several cholesterol transporters. In addition, PAR-2 activation of ex-vivo cultured vascular smooth muscle cells (VSMCs) augments their transition to a macrophage-like state (after cholesterol treatment) via upregulation of human antigen R (HuR) and resultant stabilization of the transcription factor Krüppel-like factor 4 (KLF4). Conclusion: Our results indicate PAR-2 deficiency significantly attenuates the initiation (12 weeks) and reduces the progression (24 weeks) of atherosclerosis potentially via regulation of both lipid efflux from macrophages and the phenotypic modulation of VSMCs.

Abstract 457: Transcription Coactivator MED1 Protects Against Atherosclerosis by Modulation of Macrophage Polarization

Mediator 1 (MED1), a key subunit of the Mediator complex, interacts with several nuclear receptors and transcription factors to direct gene-specific transcription. It is well-known that MED1 play important roles in lipid metabolism. However, the role and underlying mechanisms of MED1 in atherosclerosis remains unclear. Here, macrophage-specific MED1 knockout (MED1 ΔMac ) mice were generated to investigate the contribution of MED1 on atherogenesis in vivo. We demonstrate that among mice deficient in apolipoprotein E (Apoe), the additional loss of macrophage MED1 (MED1 ΔMac /ApoE -/- ) exhibited significantly larger atherosclerotic lesions in the whole aortic tree and aortic root compared with MED1 fl/fl /ApoE -/- littermates on either the chow or the western diet, and these effects were also found in low-density lipoprotein (LDL) receptor-deficient (LDLR -/- ) mice reconstituted with bone marrow from MED1 ΔMac mice. Peritoneal macrophages from MED1 ΔMac /ApoE -/- mice had significantly increased expression of gene markers for M1-like macrophages, including IL-1β, IL6,COX2, iNOS, Gro1, MCP1 and TNFα, etc, whereas decreased levels of anti-inflammatory genes for M2-like macrophages, such as Arg1, Mrc1, Retnla, Chi3l3 and PPARγ. Over-expression of MED1 using adenovirally-driven MED1 (Ad/MED1) in MED1 fl/fl /ApoE -/- macophages repressed the proinflammatory gene expression. Re-expression of MED1 using Ad/MED1 counteracted the high level of inflammatory gene in MED1 ΔMac /ApoE -/- macophages. Furthermore, gene expression profiling and PCR array showed that MED1-deficient macrophages exhibited the increased M1 targets and decreased M2 targets. These data demonstrate that MED1 expression by macrophages has anti-atherogenic effects via regulation of macrophage polarization. MED1 may be considered as a potential therapeutic target to treat atherosclerosis.

Abstract 15433: Thrombin Activation of Platelet Protease-activated Receptor 4 Augments Atherosclerosis

Objective: Platelet activation has been shown to play a critical role in both formation and propagation of atherosclerosis. Protease-activated receptors (PARs) 1 and 4 mediate signaling by the coagulation protease thrombin. While PAR1 and 4 mediate thrombin activation on human platelets, mouse platelets contain PAR4 with PAR3 acting as a cofactor. Recent studies have demonstrated direct thrombin inhibitors (DTIs) reduced atherosclerosis in hypercholesterolemic mice. In this study, we examined the effect of PAR1 and 4 deficient on atherosclerosis using low-density lipoprotein receptor deficient (Ldlr -/- ) mice. Methods and Results: Ldlr -/- mice that were PAR1 +/+ , PAR1 -/- , PAR4 +/+ , or PAR4 -/- (n = 5-20 each group) were fed a fat and cholesterol-enriched diet for 12 weeks. PAR4 deficiency attenuated aortic root (65% decrease; P = 0.001) and aortic arch (71% decrease; P = 0.001) atherosclerosis with no effects on total plasma cholesterol concentrations or lipoprotein distributions. These reductions were attributable to hematopoietic-derived PAR4 from analysis of bone marrow experiments. To determine if PAR4 mediated all effects of thrombin signaling, Ldlr -/- /PAR4 +/+ or Ldlr -/- /PAR4 -/- mice were fed a fat and cholesterol-enriched diet, supplemented with placebo or the DTI dabigatran etexilate (30 g/kg diet) for 12 weeks. Dabigatran etexilate administration reduced atherosclerosis in PAR4 +/+ mice but not in PAR4 -/- mice. PAR1 deficiency had no effect on atherosclerotic burden. Conclusion: We demonstrated whole body and hematopoietic PAR4 deficiency resulted in decreased aortic root and sinus atherosclerosis. PAR1 deficiency had no effects on atherosclerosis. Thrombin inhibition did not alter atherosclerosis in PAR4 deficient mice. Together, these results suggest that thrombin activation of PAR4 on platelets contributes to atherosclerosis.

Simvastatin prevents cardiac hypertrophy in a rat model via modulation of the JAK/STAT pathway

Simvastatin prevents cardiac hypertrophy in a rat model via modulation of the JAK/STAT pathway

CD68GFP/APOE-/- mouse: A novel model to study macrophage biology in atherosclerosis

CD68GFP/APOE-/- mouse: A novel model to study macrophage biology in atherosclerosis

Abstract 277: Diminazene Aceturate, an ACE2 Activator, Decreases AngII-Induced Atherosclerosis in Low-Density Lipoprotein Receptor--Deficient Mice

Objectives: Diminazene aceturate (DIZE) activates angiotensin converting enzyme 2 (ACE2) both in vitro and in vivo. ACE2 limits angiotensin II (AngII)/angiotensin type 1 receptor (AT1R) signaling through cleavage of AngII to form angiotensin-(1-7) (Ang-(1-7)). Previous studies in our laboratory demonstrated that whole body ACE2 deficiency increased diet-induced atherosclerosis in low density lipoprotein receptor (LDLr) deficient mice. The purpose of this study was to determine if administration of DIZE to LDLr-/- mice activates ACE2 to decrease AngII-induced atherosclerosis. Materials and Methods: Initial dose ranging studies determined the effects of 7.5, 15, or 30 mg/kg/day of DIZE infused subcutaneously by osmotic minipump for 7 days into male LDLr-/- mice. DIZE dose-dependently increased plasma Ang-(1-7) concentrations with significant elevations at 30 mg/kg/day (vehicle, 5.9 ± 0.6; DIZE, 7.9 ± 0.5 ng/mL, p<0.05). Moreover, kidney ACE2 activity increased in mice administered DIZE (30 mg/kg/day) compared to vehicle-injected controls (vehicle, 208 ± 87; DIZE, 1716 ± 424 fmol/mg/min, p<0.05). For optimal dosing, ACE2 wildtype (Ace2+/y) and deficient (Ace2-/y) LDLr-/- mice were administered either vehicle or DIZE by intramuscular injection for a total of 5 weeks. Seven days after initiation of DIZE administration, mice were infused with AngII (1,000 ng/kg/min for 28 days) to induce atherosclerosis. Compared to vehicle, administration of DIZE resulted in significant reductions in atherosclerotic lesion formation in aortic arches of AngII-infused Ace2+/y , but not Ace2-/y mice (vehicle Ace2+/y, 9.8 ± 1.2; DIZE Ace2+/y, 5.8 ± 1.2; DIZE Ace2-/y, 9.2 ± 1.8, p<0.05). Interestingly, compared to vehicle, DIZE lowered serum cholesterol concentrations in Ace2+/y but not Ace2-/y mice (vehicle Ace2+/y, 837 ± 69; DIZE Ace2+/y, 571 ± 41; DIZE Ace2-/y, 774 ± 113 mg/dl, p<0.05). Conclusions: These studies demonstrate that DIZE decreased AngII-induced atherosclerosis through an ACE2-dependent mechanism. Mechanisms for DIZE-induced reductions in AngII-induced atherosclerosis may include reductions in serum cholesterol concentrations.

Periaortic adipose tissue promotes arterial stiffness in chow fed low‐density lipoprotein receptor deficient mice (867.4)

We tested the hypothesis that young chow fed low‐density lipoprotein receptor deficient (LDLr‐/‐) mice would have periaortic adipose (PAT) related arterial stiffening, which would be associated with greater aortic remodeling. Compared with wild‐type WT controls (6mo, n=7), LDLr‐/‐ (6mo, n=7) mice had increased arterial stiffness assessed by aortic pulse wave velocity (407 ± 18 vs. 353 ± 13 cm/s, P<0.05) and intrinsic mechanical stiffness (5308 ± 623 vs. 3355 ± 330 kPa, P<0.05). Systolic (144 ± 3 vs. 146 ± 3) and diastolic (113 ± 3 vs. 115 ± 2) blood pressure did not differ between groups. Descending thoracic aortic segments from WT and LDLr‐/‐ mice were cultured with (+) or without (‐) PAT for 72 hours followed by intrinsic mechanical testing. Aortas from LDLr‐/‐ mice cultured (+) PAT (6092 ± 480 kPa, P < 0.05) had greater intrinsic mechanical stiffness compared with control (+) PAT, control (‐) PAT and LDLr‐/‐ (‐) PAT (3710 ±316, 3794 ± 496, 3473 ± 577 kPa, respectively). LDLr‐/‐ mice had reduced aortic α‐Elastin protein expression (0.6 ± 0.1 vs. 1.0 ± 0.1 AU, P<0.05), and greater advanced glycation endproducts (1.8 ± 0.2 vs. 1.0 ± 0.1 AU, P<0.05) and collagen I expressions (2.3 ± 0.2 vs. 1.0 ± 0.2 AU, P<0.05) compared with WT animals. Collectively, these data provide initial evidence for PAT in arterial stiffening, which is associated with greater aortic remodeling in chow fed LDLr‐/‐ mice.

Abstract 83: Bone Marrow Derived Cells Do Not Mediate Reductions in Cholesterol, Atherosclerosis and Adiposity in Microsomal Prostaglindin E Synthase 1 Deficient Mice Fed High Fat Diets Enriched in Cholesterol

Microsomal prostaglandin E 2 synthase-1 (mPGES-1) catalyzes the conversion of COX-2 generated PGH 2 to PGE 2 and is the predominate source of PGE 2 during and inflammatory response. We and others have demonstrated that mPGES-1 deficiency attenuates atherosclerosis in mice on a mixed background. The present study investigated the effect of mPGES-1 deficiency on atherosclerosis in C57BL/6 low density lipoprotein receptor deficient (LDLr-/-) mice. mPGES-1 deficiency attenuated atherosclerosis in LDLr-/- mice fed either a low fat (LF) (P = 0.02) or high fat (HF) (P = 0.0026) diet enriched with cholesterol, or a western diet (P = 0.02) for 17 weeks. mPGES-1 deficiency attenuated weight gain and cholesterol concentrations in mice fed a western (P = 0.004 and P < 0.05; respectively) or HF diet (P = 0.01 and P = 0.012, respectively). However, body weight and cholesterol concentrations were not different in mice fed the LF diet. These data suggest that different mechanisms mediate the reduction in atherosclerosis in mPGES-1 deficient mice fed LF and HF diets. To determine if mPGES-1 deficiency in macrophages contributed to the reduction in atherosclerosis in mice fed HF diets, 4 groups of chimeric mice were generated. Four weeks post bone marrow cell transplant (BMT) mice were fed a western diet. BMT attenuated weight gain in all groups of chimeric mice; however, weight gain was not different between any of the groups. BMT decreased atherosclerotic lesion formation 10 fold in all groups of mice. Neither bone marrow cell specific deficiency of mPGES-1 (KO>WT) or mPGES-1 specific expression in bone marrow derived cells (WT>KO) had an effect on lesion formation compared to WT>WT or KO>KO mice. Cholesterol concentrations were decreased in KO>KO and WT>KO mice compared to WT>WT (P < 0.01) and KO>WT (P< 0.05) mice. These data suggest that mPGES-1 expression in bone marrow derived cells does not contribute to the development of atherosclerosis. Moreover, these data suggest that prostanoids may play a role in hepatic cholesterol homeostasis in mice fed HF diets enriched in cholesterol thereby contributing to atherosclerotic lesion formation. Moreover, these data provide further evidence that prostanoids play a role in regulating the accumulation of diet-induced adiposity.

Abstract 565: Nuclear Receptor NR4A1 Represses SDF1a expression in macrophages and Reduces Atherosclerosis.

Rationale NR4A1 is a nuclear receptor, also known as Nur77 and is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. NR4A1-knockout (NR4A1 -/- ) mice lack Ly6C - monocytes, but have normal LyC6 + numbers. Macrophages play a critical role in atherosclerosis and we have demonstrated that NR4A1 has an anti-inflammatory function in THP-1 macrophages. The aim of the current study is to assess the function of this receptor in myeloid cells in atherosclerosis. Objective This study aims to delineate the function of Nuclear Receptor NR4A1 in macrophages in atherosclerosis. Methods and results Bone marrow-derived macrophages (BMM) from wild-type and NR4A1 -/- mice were cultured and classically activated with LPS or alternatively activated with IL4. NR4A1 -/- BMM exhibit changed expression of M2-specific markers and a pro-inflammatory polarization in response to LPS with enhanced expression of IL12, IFNγ, and SDF1α. Nitric oxide synthesis is also strongly induced in (non)-stimulated NR4A1 -/- BMM. SDF1α is a potent chemotactic factor for B cells and monocytes. The chemoattractive activity of NR4A1 -/- BMM is abolished by SDF1α inhibiting antibodies as well as by overexpression of NR4A1 in the NR4A1-deficient cells. Luciferase experiments show that NR4A1 binds directly to several response elements present in the mouse and human SDF1α promoter. A ChIP assay confirms these results. The effect of bone marrow-specific NR4A1-deficiency on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr -/- ) mice. Ldlr -/- mice with a NR4A1 -/- bone marrow develop 2.1-fold larger lesions than wild type bone marrow transplanted mice; containing more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF1α expression is also higher in these lesions, which may explain the observed aggravation of lesion formation, since SDF1α is not only a chemoattractant for monocytes but also enhances smooth muscle cell proliferation. Conclusion In macrophages the nuclear receptor NR4A1 has an anti-inflammatory function, represses SDF1α expression and, most importantly, bone-marrow transplantation studies in Ldlr -/- mice revealed that NR4A1 inhibits atherosclerosis.

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

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