ApoE KO Mice Research Articles

Atheroprotective nasal immunization with a heat shock protein 60 peptide from Porphyromonas gingivalis.

PurposeImmunization with Porphyromonas gingivalis heat shock protein 60 (PgHSP60) may have an immunoregulatory effect on atherogenesis. The aim of this study was to determine whether nasal immunization with a PgHSP60 peptide could reduce atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice.MethodsSeven-week-old male ApoE KO mice were assigned to receive a normal diet, a Western diet, a Western diet and challenge with PgHSP60-derived peptide 14 (Pep14) or peptide 19 (Pep19), or a Western diet and immunization with Pep14 or Pep19 before challenge with Pep14 or Pep19.ResultsAtherosclerotic plaques were significantly smaller in mice that received a Western diet with Pep14 nasal immunization than in mice that received a Western diet and no Pep14 immunization with or without Pep14 challenge. An immunoblot profile failed to detect serum reactivity to Pep14 in any of the study groups. Stimulation by either Pep14 or Pep19 strongly promoted the induction of CD4+CD25+forkhead box P3 (FoxP3)+ human regulatory T cells (Tregs) in vitro. However, the expression of mouse splenic CD4+CD25+FoxP3+ Tregs was lower in the Pep14-immunized mice than in the Pep14-challenged or Pep19-immunized mice. Levels of serum interferon gamma (IFN-γ) and transforming growth factor beta were higher and levels of interleukin (IL) 10 were lower in the Pep14-immunized mice than in the other groups. Induction of CD25− IL-17+ T helper 17 (Th17) cells was attenuated in the Pep14-immunized mice.ConclusionsNasal immunization with Pep14 may be a mechanism for attenuating atherogenesis by promoting the secretion of IFN-γ and/or suppressing Th17-mediated immunity.

SP-8356, a Novel Inhibitor of CD147-Cyclophilin A Interactions, Reduces Plaque Progression and Stabilizes Vulnerable Plaques in apoE-Deficient Mice.

Interactions between CD147 and cyclophilin A (CypA) promote plaque rupture that causes atherosclerosis-related cardiovascular events, such as myocardial infarction and stroke. Here, we investigated whether SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one), a novel drug, can exert therapeutic effects against plaque progression and instability through disruption of CD147-CypA interactions in apolipoprotein E-deficient (ApoE KO) mice. Immunocytochemistry and immunoprecipitation analyses were performed to assess the effects of SP-8356 on CD147-CypA interactions. Advanced plaques were induced in ApoE KO mice via partial ligation of the right carotid artery coupled with an atherogenic diet, and SP-8356 (50 mg/kg) orally administrated daily one day after carotid artery ligation for three weeks. The anti-atherosclerotic effect of SP-8356 was assessed using histological and molecular approaches. SP-8356 interfered with CD147-CypA interactions and attenuated matrix metalloproteinase-9 activation. Moreover, SP-8356 induced a decreased in atherosclerotic plaque size in ApoE KO mice and stabilized plaque vulnerability by reducing the necrotic lipid core, suppressing macrophage infiltration, and enhancing fibrous cap thickness through increasing the content of vascular smooth muscle cells. SP-8356 exerts remarkable anti-atherosclerotic effects by suppressing plaque development and improving plaque stability through inhibiting CD147-CypA interactions. Our novel findings support the potential utility of SP-8356 as a therapeutic agent for atherosclerotic plaque.

Deletion of the Mir-106b~\u200925 MicroRNA cluster attenuates atherosclerosis in Apolipoprotein E knockout mice

BackgroundMicroRNAs are short non-coding RNAs that regulate gene expression. The aim of this study was to gain an understanding of the possible role of the miR-106b~ 25 microRNA cluster in regulating atherosclerosis in mice.MethodsMiR-106b~ 25 knockout mice were outcrossed into Apolipoprotein E (ApoE) knockout background to generate double knockout mice. At 36 weeks of age, lesion size was evaluated in the aortic sinus by oil-red-O staining.ResultsLesion size was 2-fold smaller in double KO mice in comparison to ApoE KO mice. In addition, collagen staining showed a trend towards a stable plaque phenotype in the double KO mice. Lipid profiling of plasma samples of double KO and ApoE KO mice using FPLC revealed over 2-fold decrease in Very low density lipoprotein (VLDL) cholesterol content and a 50% decrease in low density lipoprotein (LDL) cholesterol content in double KO mice. By using target prediction software, we have identified several possible targets for the miR-106b~ 25 cluster including the VLDL and LDL receptors. We found that upon feeding miR-106b~ 25 KO mice with high fat diet, the expression of LDL and VLDL receptors was higher than in the wild-type mice, suggesting the miR-106b~ 25 cluster regulates atherosclerosis by influencing clearance of VLDL and LDL from the plasma.ConclusionsWe identified the miR-106b~ 25 cluster as a novel regulator of atherosclerosis in ApoE KO mice, presumably by regulating plasma cholesterol levels.

Varenicline aggravates atherosclerotic plaque formation in nicotine-pretreated ApoE knockout mice due to enhanced oxLDL uptake by macrophages through downregulation of ABCA1 and ABCG1 expression

Varenicline is a widely used and effective drug for smoking cessation. We previously reported that varenicline aggravates atherosclerosis in apolipoprotein E knockout (ApoE KO) mice. However, it remains unknown whether varenicline affects cardiovascular events in patients with nicotine addiction. Here, we examined the effect of varenicline on atherosclerotic plaque formation in nicotine-pretreated ApoE KO mice and oxidized low-density lipoprotein (oxLDL) uptake in nicotine-treated peritoneal macrophages. Varenicline caused significant progression of plaque formation in the whole aorta and aortic root and further accelerated the increased formation of a macrophage-rich plaque area in the aortic root in nicotine-pretreated ApoE KO mice. Varenicline (10 μM) enhanced oxLDL uptake in peritoneal macrophages. Furthermore, this treatment significantly further lowered the decreased protein levels of ATP-binding cassette (ABC) transporter without affecting the expression of scavenger receptors LOX-1 and CD36 in RAW264.7 cells treated with 100 nM nicotine. Varenicline enhanced nicotine-induced oxLDL uptake in macrophages through decreased expression of cholesterol efflux transporters ABCA1 and ABCG1 and thereby progressed atherosclerotic plaque formation. Taken together, we tentatively conclude that nicotine exposure before and/or during varenicline treatment can aggravate varenicline-increased atherosclerotic plaque formation and progression. Therefore, this enhanced risk requires special consideration when prescribing varenicline to smoker patients.

The active components derived from Penthorum chinensePursh protect against oxidative-stress-induced vascular injury via autophagy induction

Oxidative stress-induced damage has been proposed as a major risk factor for cardiovascular disease and is a pathogenic feature of atherosclerosis. Although autophagy was reported to have a protective effect against atherosclerosis, its mechanism for reducing oxidative stress remains un-elucidated. In this study, we have identified 4 novel autophagic compounds from traditional Chinese medicines (TCMs), which activated the AMPK mediated autophagy pathway for the recovery of mitochondrial membrane potential (MMP) to reduce the production of reactive oxygen species (ROS) in Human umbilical vein endothelial cells (HUVECs). In this study, 4 compounds (TA, PG, TB and PG1) identified from Penthorum chinense Pursh (PCP) were demonstrated for the first time to possess binding affinity to HUVECs cell membranes via cell membrane chromatography (CMC) accompanied by UHPLC-TOF-MS analysis, and the 4 identified compounds induce autophagy in HUVECs. Among the 4 autophagic activators identified from PCP, TA (Thonningianin A, Pinocembrin dihydrochalcone-7-O-[3″-O-galloyl-4″,6″-hexahydroxydiphenoyl]-glucoside) is the major chemcial component in PCP, which possesses the most potent autophagy effect via a Ca2+/AMPK-dependent and mTOR-independent pathways. Moreover, TA efficiently reduced the level of ROS in HUVECs induced by H2O2. Additionally, the expression of pro- and cleaved-IL-1β in the aortic artery of ApoE-KO mice were also alleviated at the transcription and post-transcription levels after the administration of TA, which might be correlated to the reduction of oxidative-stress induced inflammasome-related Nod-like receptor protein3 (NLRP3) in the aortic arteries of ApoE-KO mice. This study has pinpointed the novel autophagic role of TA in alleviating the oxidative stress of HUVECs and aortic artery of ApoE-KO mice, and provided insight into the therapeutic application of TA in treatment of atherosclerosis or other cardiovascular diseases.

1440Anti-inflammatory adipokine, omentin, attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein-E knockout mice

Abstract Background Abdominal aortic aneurysm (AAA) is an increasing and life-threatening disease. Obesity is associated with an increased risk of AAA. Omentin is a circulating adipokine, which is downregulated by obesity. Recently we have demonstrated that omentin is an anti-inflammatory adipokine that prevents the development of atherosclerosis in apolipoprotein-E knockout (apoE-KO) mice. Here we examined whether omentin could modulate angiotensin II-induced AAA formation in apoE-KO mice. Methods and results To overexpress human omentin in apoE-KO mice, apoE-KO mice were crossed with transgenic mice expressing the human omentin gene in fat tissue under the control of AP2 promoter (OMT-Tg mice). Circulating levels of human omentin in apoE-KO/OMT-Tg mice were approximately threefold higher than those in healthy human subjects, whereas human omentin was undetectable in apoE-KO mice. There were no differences in body weight, blood pressure and heart rate between apoE-KO/OMT-Tg and apoE-KO mice. We also subjected apoE-KO/OMT-Tg and apoE-KO mice at 24 weeks of age to continuous angiotensin II-infusion by using osmotic mini pumps for 4 weeks, which is a widely-accepted model of experimental AAA. ApoE-KO/OMT-Tg mice exhibited a lower incidence of AAA formation and a reduced maximal diameter of AAA determined by direct measurement and ultrasound imaging as compared with apo-E KO mice. In histological analyses with van Gieson staining, apoE-KO/OMT-Tg mice showed attenuated disruption of medial elastic fibers in response to angiotensin II compared with apo-E KO mice. ApoE-KO/OMT-Tg mice also displayed reduced mRNA levels of matrix metalloproteinase (MMP) 2 and MMP9 as well as pro-inflammation genes including interleukin (IL)-6 in aortic walls compared with apo-E KO mice. Treatment of human monocyte-derived macrophages with human omentin protein attenuated LPS-stimulated expression of MMP9, TNF-α and IL-6. Omentin treatment also reduced LPS-induced activation of MMP9 in cultured media of human macrophages as evaluated by gelatinolytic zymography. Omentin treatment increased phosphorylation levels of Akt in human macrophages. The suppressive effects of omentin on inflammatory response in macrophages were reversed by treatment with LY294002, which is an inhibitor of PI3 kinase/Akt signaling. Conclusion These data suggest that omentin acts as an adipokine that can attenuate angiotensin II-induced development of AAA through suppression of MMP activation and inflammatory response in the vascular wall.

3306Endothelial cell senescence accelerates atherosclerosis by enhancing monocyte recruitment via hyper-reactivity to inflammatory stimuli

Abstract Background Ageing is a significant risk factor for atherosclerotic diseases. Vascular senescence has been considered to play an important role in the progression of atherosclerosis; however, it remains unclear whether endothelial cell (EC) senescence is causally involved in the pathogenesis of atherosclerosis. Purpose The purpose of this study is to elucidate a causative role of senescent EC in the progression of atherosclerosis. Methods Telomeric repeat-binding factor 2 (TRF2) plays a central role in telomere maintenance and protection against end-to-end fusion of chromosome. We previously reported that overexpression of TRF2-dominant negative mutant (TRF2DN) induced premature senescence in EC. We recently generated EC-specific progeroid mice by overexpressing TRF2DN specifically in EC (TRF2DN-Tg), and then generated ApoE-KO/TRF2DN-Tg mice to analyze a role of EC senescence in atherosclerosis. These mice were fed with a high cholesterol-diet, and atherosclerosis was assessed by en face analysis of whole aorta and histological analysis of aortic sinus. In vitro studies to analyze the underlying mechanisms were performed using replicative senescent HUVECs. Results En face analysis of aorta revealed that atherosclerotic lesions were significantly increased in ApoE-KO/TRF2DN-Tg mice comparing with that in ApoE-KO mice at as early as 2 weeks after high-cholesterol diet. Histological analysis of aortic sinus also exhibited accelerated atherosclerosis in ApoE-KO/TRF2DN-Tg mice in association with increased macrophage infiltration. Mechanistically, we found that the induction of adhesion molecules such as VCAM-1, ICAM-1, and E-selectin in response to low-grade inflammatory stimuli was substantially augmented in senescent ECs comparing with that in young ECs. As a result, monocyte adhesion was significantly enhanced in senescent ECs. Of note, eNOS inhibition did not affect the hyper-reactivity of senescent EC to inflammation, while antagonizing NF-kB abolished it. Nuclear translocation of NF-kB in response to inflammation was not different between young and senescent ECs, suggesting that NF-kB transcriptional activity might be enhanced in senescent ECs. Conclusion We revealed a causative role of EC senescence in the progression of atherosclerosis in vivo using unique EC-specific progeroid mice. Our findings revealed that EC senescence is a bona fide risk for atherosclerosis, and thus senescent ECs are attracting pharmacotherapeutic targets for the prevention and/or treatment of atherosclerotic disease in elderly population.

5219A novel senolytic drug, seno-7284 ameliorates age-related cardiometabolic diseases

Abstract Background Senescence at cellular level develops with various genotoxic stresses and it plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so called “senolysis” has potential to become a next generation therapy for age-related disorders including cardiovascular diseases, pulmonary emphysema, Alzheimer's diseases, etc. However, currently there is no senolytic agent available in clinical settings. Purpose Present study was aimed to identify a novel senolytic agent effective for cardiometabolic diseases in compounds already available in clinical settings. Here we demonstrate a compound called “seno-7284” exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis and progeroid. Methods We generated 1) diet-induced obase and diabetic model by imposing a high fat diet for two months, 2) atherosclerosis mice model by imposing western diet to ApoE homozygous knockout mice (ApoE-KO mice) for three months, and 3) Zmpste24 homozygous knockout mice (Zmpste24-KO mice) as a progeroid mice model. We administrated seno-7284 by mixing it into the diet (0.03% w/w). In one, two or four weeks after the administration of seno-7284 to each mice model, we collected tissue samples for further analyses. Results Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of dietary obese mice as senescence-associated beta-galactosidase (SA-beta-gal) staining exhibits (Figure a). This effect was associated with the suppression in systemic glucose intolerance (Figure b), and adipose tissue inflammation in four weeks after the administration of seno-7284. Administrating seno-7284 for two weeks also reduced accumulation of senescent cells in atherosclerotic lesion in aorta of ApoE-KO mice (Figure c), and inhibited the progression of atherosclerosis (Figure d). Surprisingly, this drug significantly improved the lifespan of Zmpste24-KO mice by administering it from 12 weeks old. Further analysis including RNA-seq or metabolomic analysis suggested that seno-7284 stimulates endogenous senolytic function of NK cells and CD8+ T cells. Conclusion Our results indicate that seno-7284 mediates its biological effects by inducing senolysis in some murine aging models. Seno-7284 would become a promising therapeutic agent for age-related cardiometabolic diseases.

P723A novel CD147 inhibitor SP-8356 attenuates plaque progression and stabilizes vulnerable plaque in ApoE-deficient mice

Abstract Aim Plaque vulnerability is the crucial pathophysiological feature in atherosclerosis-related cardiovascular event such as myocardial infarction and stroke. CD147 has been suggested to play key roles in plaque vulnerability through interacting with cyclophilin A (CypA) and resultant activation of matrix metalloproteinase-9 (MMP-9). Here we report that the novel synthetic CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) inhibits CD147/MMP-9 pathways and reduces plaque progression and stabilizes plaque vulnerability. Methods Advanced atherosclerotic plaque was induced in apolipoprotein E-deficient (ApoE KO) mice by partial ligation of the right carotid artery coupled with an atherogenic diet. SP-8356 (50 mg/kg) was orally given daily for 3 weeks. Histomolecular analysis was carried out on harvested carotid arteries. Results Surface plasmon resonance assay showed the specific binding of SP-8356 with CD147. SP-8356 inhibited CypA-CD147 interaction and MMP-9 activation. In ApoE KO mice model, SP-8356 inhibited plaque formation, reduced the number of macrophages, increased the number of vascular smooth muscle cells, increased the fibrous cap thickness, and increased the collagen type I contents in fibrous cap. SP-8356 also reduced the apoptotic cells in the plaque lesion. Conclusions Owing to its improvement of plaque stability and inhibitory effect on plaque development, SP-8356 could be a potential therapeutic drug candidate for atherosclerosis and related clinical manifestations.

5892Elimination of cells expressing Senescence associated glycoprotein (SAGP) attenuates the atherosclerotic diseases

Abstract Cellular senescence is defined as a state of irreversible growth arrest and is accompanied by changes of both cell morphology and gene expression. Although accumulation of senescent vascular endothelial cells impair the vessel homeostasis and promote atherosclerotic diseases, underlying mechanisms are largely unknown. In this study, we identified a novel protein, senescence-associated glycoprotein (SAGP), as a biomarker of cellular senescence and we found modulation of SAGP or elimination of senescent cells targeting SAGP would become a novel therapy for atherosclerotic diseases. We found that SAGP expression was significantly increased in human endothelial cells undergoing replicative senescence compared with young endothelial cells. We also found SAGP expression in aorta was significantly increased both in chronological aging mice or ApoE knockout mice. Furthermore, we measured SAGP expression in patients registered in our hospital and found that mean SAGP expression was significantly higher in patients with atherosclerotic diseases compared to patients without atherosclerotic diseases.These data suggest that SAGP would become a novel cellular senescence and/or atherosclerotic disease marker. Genetic deletion of SAGP resulted in high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. And this associated with suppression of mitochondrial autophagy, mitophagy. We found SAGP co-localized with lysosome by immunocytochemistry. In addition, the electron microscopy analysis revealed that the dysfunctional lysosomes were accumulated in SAGP knockdown endothelial cell, suggesting that SAGP maintain lysosomal homeostasis. Next, wegenerated ApoE-KO/ SAGP overexpression mice and found that atherosclerotic plaque burden was attenuated in these double-transgenic mice. In contrast, SAGP/ApoE double knockout mice showed progression in atherosclerosis. These data suggest that modulation of SAGPwould become a new therapeutic target for atherosclerotic diseases. SAGP vaccine Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes and also extended the lifespan. We have taken another approach for atherosclerotic diseases, senolytic therapy targeting SAGP. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice, in which we could eliminate the SAGP- positive senescent cells using DT (diphtheria toxin). We found elimination of SAGP positive senescent cells significantly reduced the atherosclerotic plaque burden, indicating that SAGP would become a useful target for senolytic therapy. We then developed a cytotoxic vaccine targeting SAGP. Treatment with SAGP vaccine successfully eliminated SAGP positive senescent cells. Administration of SAGP vaccine to ApoE-KO mice significantly reduced atherogenesis. These data indicate that targeting SAGP-positive cells could become a strategy for senolytic therapy.

Abstract 124: NaCl Treatment Prior To Chronic Angiotensin Ii Infusion Exaggerates Vascular Inflammation And The Development of Abdominal Aortic Aneurysms By Affecting Immune Cell Response

Chronic high salt (NaCl) intake is a major risk factor for cardiovascular diseases. Recent studies showed that excessive chronic NaCl intake affects immune response and thereby contributes to the pathogenesis of vascular injury. However, it remains unclear whether NaCl intake initially primes immune cell response by promoting memory T cells and sensitize the development of vascular injury in response to a subsequent stimulus. Therefore, we investigate whether 14 days of NaCl treatment (1% NaCl) followed by a 7 day washout period affects the incidence and outcome of abdominal aortic aneurysms (AAA) in apolipoprotein E deficient (ApoE-KO) mice infused chronically with angiotensin II (Ang II) (1000ng/kg/min) via osmotic minipumps for 10 days. Assessment of AAA was done by magnetic resonance imaging (MRI). To visualize vascular inflammation in vivo , perfluorocarbon nano-emulsions (PFC) injected intravenously before and 2, 4, 7 and 10 days after chronic Ang II infusion were detected by combining 1H/19F MRI using a 19 F RARE sequence. After 10 days of Ang II infusion, aortas with perivascular fat were dissected. Aortic inflammation and immune cells infiltration were analyzed by flow cytometery, immunohistology and realtime PCR. The survival rate of ApoE-KO mice pretreated with NaCl was significant lower and the AAA incidence rate was significant higher compared to non-NaCl treated apoE-KO mice. The amount of 19 F signal detected in areas of AAA was significantly increased in apoE-KO mice pretreated with NaCl. Histological analysis confirmed these results. At day 10, proinflammatory cytokines like TNF-alpha, iNOS and TGF-ß as well as numbers of neutrophils, monocytes as well as effector memory CD4 and CD8 T cells were significantly higher in aortas of NaCl pretreated apoE-KO mice compared to non-NaCl treated mice. In conclusion, these results suggest a significant role of NaCl in priming immune cell phenotype and thereby mediating vascular inflammation and damage in Ang II infused apoE-KO mice.

Abstract 498: The Effects of Low-carbohydrate High-fat Diet on Vascular Remodeling

Background: Low carbohydrate diet (LCD) has been reported to reduce body weight and cardiovascular risk factors, including diabetic mellitus and dyslipidemia. Therefore, LCD was once expected to prevent cardiovascular events. However, recent meta-analysis reported that LCD was related to an increase in cardiovascular events. Moreover, other cohort studies reported that higher all-cause and cardiovascular mortality were observed only in LCD with animal derived-fat (LCD-A), not in LCD with plant derived-fat (LCD-P). Precise mechanisms of these effects have not been clarified, so we explored the potential roles of LCD-A and LCD-P in blood vessel remodeling by using two types of vascular disease model mice, atherosclerotic model and vascular injury model. Method: We generated two types of LCDs, beef tallow-based LCD (LCD-A) and cacao butter-based LCD (LCD-P). Six-week-old male ApoE Knockout (ApoE KO) mice (atherosclerosis model mice) and wild-type mice were subjected to LCD-A, LCD-P or normal chow for 14 weeks, and %plaque area of aortic root and serum lipid profile were measured. For vascular injury model, mice were inserted with a wire in a femoral artery of male 10-week-old wild-type mice and subjected to the three-types chow. After 4 weeks, femoral arteries were excised and embedded in paraffin and assessed with the extent of vascular injury severity. The femoral artery without injury on the other side was served as control. Results: In ApoE KO mice, serum levels of total cholesterol (TC) and triglyceride (TG) were decreased in LCD group (TC: p<0.01, TG: p<0.05 vs normal chow), but there was no significant difference between LCD-A to LCD-P. In wild-type mice, serum TC level was increased in LCD-A group (p<0.0001 vs normal chow). LCD had no effect on %plaque area of aortic root in ApoE KO mice. In vascular injury model mice, there was no significant difference in the extent of neointimal hyperplasia induced by wire-injury among groups. Conclusion: Our preliminary data showed that LCD had no effect on vascular remodeling despite the presence of significant alteration in lipid profiles. Additional experiments are needed to confirm the results and clarify the precise mechanisms.

Abstract 285: Oxidized Low Density Lipoprotein and Angiotensin II Causes Vascular Senescence Via AT1R Signal-mediate Mitochondrial Fission

Introduction: Metabolic stress including oxidized low density lipoprotein (ox-LDL) and angiotensin II (AngII) cause mitochondrial dysfunction and vascular senescence. Mitochondria are highly dynamic organelles that constantly change their morphology to fusion or fission. This study aims to clarify how mitochondrial dynamics are involved in the etiology of vascular senescence. Methods: VSMC and HUVEC were stimulated by either ox-LDL or AngII. We also conducted in vivo experiment using C57BL6 (WT), apolipoprotein E (ApoE) deficient and the double knockout of ApoE and AT1R (DKO) mice. Results: Either ox-LDL or AngII induced excessive mitochondrial fission though phosphorylation of Drp1 at Ser616, mitochondrial dysfunction assessed by JC1, reactive oxygen species production measured with Mito-Sox Red and Amplex assay and cellular senescence analyzed by senescence associated beta galactosidase staining and immunoblot of p53 and p21 in cells. Administration of mdivi-1, a specific inhibitor of Drp1, restored imbalance of mitochondrial dynamics and these detrimental alterations. Treatment with angiotensin II type1 receptor (AT1R) inhibitor also inactivated Drp1 and improved imbalance of mitochondrial dynamics, mitochondrial dysfunction and cellular senescence. These results suggest that ox-LDL- or AngII-induced mitochondrial dysfunction and cellular senescence were derived from Drp1-dependent mitochondrial fission. Administration of AT1R inhibitor to HUVEC or SMC with ox-LDL prevented cells against excessive mitochondrial fission, its dysfunction and cellular senescence. The degree of vascular senescence was higher, the number of fused mitochondria and mitochondrial function were lower and mitochondrial oxidative stress were higher in either C57BL6 with AngII infusion or ApoE KO mice than those of control or WT mice, respectively. Treatment with mdivi-1 to these mice reduced mitochondrial fission, oxidative stress and attenuated vascular senescence. The number of fused mitochondria and its function were higher and the degree of vascular senescence were lower in DKO than those in ApoE KO mice. Conclusion: Either ox-LDL or AngIIcauses cellular and vascular senescence through AT1R signal-mediated mitochondrial fission.

Abstract 746: The Effect of Aging-related Metabolic Disorder on Resistin Expression and Valvular Heart Disorder

Calcific aortic valve stenosis (AS) is the most prevalent valvular heart disease and the third most frequent cardiovascular disease after coronary artery disease and hypertension. AS is characterized by a period of asymptomatic progressive valve calcification, which eventually leads to the development of a severe stenosis and to the onset of symptoms. Visceral obesity is a strong predictor of type-2 diabetes and is associated with insulin resistance. In addition, increased visceral adiposity leads to an increase in systemic release in resistin (Retn) and possibly interleukins. Elevation of circulating adipokines such as Retn plays a role in the development of muscle insulin resistance. Moreover, higher Retn levels were associated with increased valvular inflammation and calcification in the elderly patients, whereas this association was not observed among middle-aged patients. In the current study, we explored the potential role of Retn in ectopic valvular calcification. Analyses of scanning electron microscopy micrographs of calcified aortic valve showed that Retn is more abundant next to calcified nodes. These data corroborate with increased levels of circulating Retn in apoE mice that were fed a high fat high sucrose diet for 24 weeks. Moreover, apoE-/-Retn-/- double KO mutant mice have lower plasma cholesterol level compared to apoE KO mice. In addition, knocking down Retn decreased valve lipid infiltration, lowered glycaemia, calcium nodes and reduced fibrosis. In conclusion, Retn may be a new pharmacological target to stop the progression of aortic stenosis in patients with visceral obesity.

Abstract 804: Elimination of Cells Expressing Senescence Associated Glycoprotein Attenuates Theatherosclerotic Diseases

Although accumulation of senescent vascular endothelial cells impairs the vessel homeostasis and promote atherosclerotic diseases, underlying mechanisms are largely unknown. We found that Senescence associated glycoprotein (SAGP) expression was significantly increased in human endothelial cells undergoing replicative senescence. SAGP expression in aorta was significantly increased in Apo-E knockout mice. Furthermore, mean SAGP expression was significantly higher in the leukocytes of the patients with atherosclerotic diseases.These data suggest that SAGP would become the novel cellular senescence and/or atherosclerotic disease marker. The genetic deletion of SAGP resulted in high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. And this associated with suppression of mitochondrial autophagy, mitophagy, suggesting that SAGP protect cells from ROS by inducing mitophagy. Thus, wegenerated ApoE-KO / SAGP gain-of-function transgenic mice and found that atherosclerotic plaque burden was attenuated in these transgenic mice. In contrast, in SAGP / ApoE double knockout mice atherosclerosis were impaired. These data suggest that modulation of SAGPwould become a new therapeutic target for atherosclerotic diseases.Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice,inducible elimination of SAGP positive senescent cells upon administration of diphtheria toxin. Elimination of SAGP expressing cells significantly reduced the atherosclerotic plaque burden. Furthermore, we developed a cytotoxic vaccine targeting SAGP. Administration of SAGP vaccine to ApoE-KO mice significantly reduced atherogenesis. These data indicate that targeting SAGP-positive cells could be a strategy for senolytic therapy.

The flagellin-TLR5-Nox4 axis promotes the migration of smooth muscle cells in atherosclerosis

We hypothesized that NADPH oxidase 4 (Nox4) is involved in the formation of neointimal atherosclerotic plaques through the migration of smooth muscle cells (SMCs) in response to flagellin. Here, we demonstrate that TLR5-mediated Nox4 activation regulates the migration of SMCs, leading to neointimal plaque formation in atherosclerosis. To investigate the molecular mechanism by which the TLR5-Nox4 cascade mediates SMC migration, we analyzed the signaling cascade in primary vascular SMCs (VSMCs) from wild-type (WT) or Nox4 KO mice. Stimulation of VSMCs from Nox4 KO mice with flagellin failed to induce H2O2 production and Rac activation compared with stimulation of VSMCs from WT mice. Moreover, the migration of Nox4-deficient VSMCs was attenuated in response to flagellin in transwell migration and wound healing assays. Finally, we performed partial carotid artery ligation in ApoE KO and Nox4ApoE DKO mice fed a high-fat diet (HFD) with or without recombinant FliC (rFliC) injection. Injection of rFliC into ApoE KO mice fed a HFD resulted in significantly increased SMC migration into the intimal layer, whereas SMC accumulation was not detected in Nox4ApoE DKO mice. We conclude that activation of the TLR5-Nox4 cascade plays an important role in the formation of neointimal atherosclerotic plaques.

A NOVEL CD147 INHIBITOR SP-8356 ATTENUATES PLAQUE PROGRESSION AND STABILIZES VULNERABLE PLAQUE IN APOE-DEFICIENT MICE

Objective: Plaque vulnerability is the crucial pathophysiological feature in atherosclerosis-related cardiovascular event such as myocardial infarction and stroke. CD147 has been suggested to play key roles in plaque vulnerability through interacting with cyclophilin A (CypA) and resultant activation of matrix metalloproteinase-9 (MMP-9). Here we report that the novel synthetic CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) inhibits CD147/MMP-9 pathways and reduces plaque progression and stabilizes plaque vulnerability. Design and method: Advanced atherosclerotic plaque was induced in apolipoprotein E-deficient (ApoE KO) mice by partial ligation of the right carotid artery coupled with an atherogenic diet. SP-8356 (50 mg/kg) was orally given daily for 3 weeks. Histomolecular analysis was carried out on harvested carotid arteries. Results: Surface plasmon resonance assay showed the specific binding of SP-8356 with CD147. SP-8356 inhibited CypA-CD147 interaction and MMP-9 activation. In ApoE KO mice model, SP-8356 inhibited plaque formation, reduced the number of macrophages, increased the number of vascular smooth muscle cells, increased the fibrous cap thickness, and increased the collagen type I contents in fibrous cap. SP-8356 also reduced the apoptotic cells in the plaque lesion. Conclusions: Owing to its improvement of plaque stability and inhibitory effect on plaque development, SP-8356 could be a potential therapeutic drug candidate for atherosclerosis and related clinical manifestations.

483-P: Pioglitazone Suppresses Macrophage Proliferation in Apolipoprotein-e Deficient Mice by Activating PPARγ

Background and Aims: Local macrophage proliferation in the atherosclerotic plaque is linked to the enhanced atherosclerosis progression. Although we reported that troglitazone, a thiazolidinedione (TZD), suppressed oxidized low-density lipoprotein (Ox-LDL)-induced macrophage proliferation, its mechanisms have been unclear. We therefore investigated the mechanism(s) of the inhibitory effect of macrophage proliferation by another TZD, pioglitazone. Methods: High-fat diet (HFD)-fed, apolipoprotein E-deficient (ApoE KO) mice were treated orally with pioglitazone (10 mg/kg/day). After 8 weeks of treatment, the whole aorta or frozen sections of the aortic sinus were stained with Oil red O. To identify macrophage proliferation and apoptosis, we performed Ki67/Iba-1 double immunohistochemical staining and TUNEL method, respectively. Results: Atherosclerosis progression was suppressed in aortic sinuses of pioglitazone-treated ApoE KO mice, and they showed fewer proliferating macrophages in the plaques. Pioglitazone suppressed Ox-LDL-induced macrophage proliferation in a dose-dependent manner, which was ameliorated by peroxisome proliferator-activated receptor-γ (PPARγ) siRNA. Low concentrations (less than 100 µM) of pioglitazone, which can suppress macrophage proliferation, activated PPARγ in macrophages, but did not induce macrophage apoptosis. Pioglitazone did not induce TUNEL-positive cells in atherosclerotic plaques in ApoE KO mice. Conclusions: Pioglitazone suppresses macrophage proliferation through PPARγ without inducing macrophage apoptosis. These findings imply that pioglitazone could prevent macrovascular complications in diabetics. Disclosure S. Murakami-Nishida: None. T. Matsumura: None. S. Nishida: None. T. Senokuchi: None. N. Ishii: None. S. Yamada: None. Y. Morita: None. T. Wada: None. H. Motoshima: None. T. Kondo: None. E. Araki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Merck & Co., Inc., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi.

Mild Hyperhomocysteinemia Induced by a Hypomethylating Diet Does Not Favor Aortic Plaque Formation in apoE Knockout Mice (P24-037-19)

ObjectivesHyperhomocysteinemia is an independent risk factor for atherosclerosis and cardiovascular disease through mechanisms still incompletely defined. We investigated the impact of mild diet-induced accumulation of homocysteine on atherosclerotic plaque formation in apoE knockout (KO) mice, a model for atherosclerosis in humans. We hypothesize that diet induced hyperhomocysteinemia will promote plaque development. Methods7 wk-old male apoE-KO mice (n = 5) were fed a methyl-deficient diet for 16 wk. The diet consisted of a purified high (40%)-fat high-methionine diet with restricted levels of B vitamins and choline (HypoMet). A second group of animals (control, n = 5) was fed a normal-methyl matched diet. After 4 and 12 wk, plasma homocysteine was quantified by reverse phase HPLC with fluorometric detection, and a panel of inflammatory cytokines (MCP-1; TNF-; IL-17A/F; IL-2; IL-6, IL-10; KC/GRO; MIP-1; IFN-) were assayed (U-plex, Meso Scale Discovery). After 16 wk, mice were euthanized and perfusion-fixed aortas were stained for lipids (Oil Red-O) and subjected to 2-D-quantification of stained plaque (Image J software), and to 3-D analysis by magnetic resonance imaging (MRI, Agilent 14-tesla microimaging system). Standard 3-D gradient echo imaging yielded a resolution of 20 microns isotropic. Data were reconstructed using Matlab and segmented to obtain plaque volumes using Avizo 9.0. ResultsHypoMet-mice had significantly higher plasma homocysteine (μM), when compared to controls, at 4 wk (10.6 ± 4.5 vs 2.3 ± 0.8, P < 0.05) and 12 wk (7.6 ± 1.5 vs 2.5 ± 1.1, P < 0.05). No significant differences were observed in inflammatory cytokines. Surprisingly, Oil Red-O staining revealed that HypoMet-mice did not display more plaque coverage (37 ± 6.9%) than controls (54.4 ± 10.4%). 14-T MRI results (Fig.1) confirmed that HypoMet-mice did not present higher plaque volumes than controls (0.61 ± 0.18 mm3 vs 1.2 ± 0.35 mm3, for HypoMet mice vs controls). ConclusionsA mild accumulation of homocysteine, induced by a methyl-deficient diet, did not favor atherosclerosis formation in the aortas of apoE-KO mice. Future analysis of intracellular hypomethylation may explain the observed effects of mild hyperhomocysteinemia on plaque formation. Funding SourcesGraduate Program and University Funding. Supporting Tables, Images and/or Graphs▪

482-P: Linagliptin Induces M2 Macrophage Polarization and Suppresses Progression of Atherosclerosis in High-Fat-Fed ApoE-Deficient Mice

Background: Dipeptidyl peptidase-4 (DPP-4) inhibitors suppressed the progression of atherosclerosis in atherosclerotic mouse models. However, exact mechanism has been unclear. In this study, we investigated whether linagliptin suppressed progression of atherosclerosis in high fat diet (HFD)-fed apolipoprotein-E-deficient (ApoE KO) mice via anti-inflammatory effects on macrophages. Methods: HFD-fed ApoE KO mice were treated orally with linagliptin (10 mg/kg/day). After 8 weeks of treatment, whole aorta and aortic sinus were stained with Oil red O, or anti-4-Hydroxynonenal (4-HNE) and anti-F4/80 antibodies. Mouse bone marrow-derived macrophages were used in vitro study. Intracellular ROS generation was evaluated by H2DCF-DA assay. Expression of iNOS, MCP-1, TNF-α, arginase-1, Mgl2 and Ym1 mRNA, and macrophage polarization were evaluated by real-time RT-PCR and FACS scan, respectively. Results: There were no significant differences on food intake, body weight, blood glucose levels between each group, but cholesterol concentrations in VLDL and LDL particle were lower in linagliptin group. Oil-Red-O staining demonstrated that size of atherosclerotic lesions was decreased in linagliptin group. The 4-HNE-positive area and number of macrophages in plaques were decreased, and mRNA expression of arginase-1, Mgl2 and Ym1 in the aorta were higher in linagliptin groups than those in control groups. Linagliptin suppressed ROS generation, increased mRNA expression of arginase-1, Mgl2 and Ym1, and ameliorated LPS+IFNγ-induced reduction of M2 macrophages. Conclusion: This study suggests that linagliptin suppresses the progression of atherosclerosis in HFD-fed apoE KO mice. Moreover, linagliptin suppresses ROS generation and increases the polarization of M2 macrophages. These findings may indicate the beneficial effects of linagliptin for the prevention of diabetic macrovascular complications. Disclosure S. Nishida: None. T. Matsumura: None. T. Senokuchi: None. N. Ishii: None. S. Murakami-Nishida: None. S. Yamada: None. Y. Morita: None. T. Wada: None. H. Motoshima: None. T. Kondo: None. E. Araki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Merck &amp; Co., Inc., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi.