ApoE KO Research Articles

Securinine protects vascular dysfunction targeting atherosclerosis in vascular endothelial cells, smooth muscle cells, and apolipoprotein E deficient mice

Atherosclerosis is a chronic vascular disease and characterized by accumulation within the intima of inflammatory cells, smooth muscle cells, lipid, and connective tissue. Here, we suggest securinine as an anti‐atherosclerotic agent to attenuate vascular dysfunction and lesion. To identify natural agents that commonly reverse advanced atherosclerotic plaque to early atherosclerotic plaque, significantly regulated genes were analyzed in silico. The differentially expressed genes from 9 intimal thickening and 8 fibrous cap atheroma tissue which were collected from GEO data were assessed by the connectivity map. Natural candidate securinine, a main compound from Securinega suffruticosa, was selected and administrated 1, 5 mg/kg/day in apolipoprotein‐E‐deficient (ApoE KO) mice for 18 weeks. Securinine significantly showed lowered blood pressure and improvement of metabolic parameters with hyperlipidemia. The impairment in vasorelaxation was remarkably decreased by treatment with securinine. H&E staining revealed that treatment with securinine reduced atherosclerotic lesions. Securinine suppressed the expression of adhesion molecules and matrix metalloproteinase‐2/‐9 in both ApoE KO and vascular endothelial cells (HUVEC). In HUVEC pretreatment with securinine significantly inhibited ROS generation and NF‐κB activation. Growth curve assays using the real‐time cell analyzer showed that securinine significantly decreased TNF‐α‐induced aortic smooth muscle cell proliferation and migration in a dose‐dependent manner. Thus securinine may be a potential natural candidate for the treatment of atherosclerosis because it attenuates vascular inflammation and dysfunction as well as vascular lesion.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2017R1A5A2015805)(2017R1D1A3B03029089) (2019R1A2C1085650).

A Novel Small Molecule Compound SP-8356 Targeting CD147 Suppresses Plaque Progression and Promotes Plaque Stability in ApoE-Deficient Mice

Background: Plaque vulnerability is the important therapeutic target in atherosclerosis and its related cardiovascular disease. 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 cardiovascular diseases.

Hesperidin blocks varenicline-aggravated atherosclerotic plaque formation in apolipoprotein E knockout mice by downregulating net uptake of oxidized low-density lipoprotein in macrophages

Varenicline is a widely used and effective drug for smoking cessation. We have previously reported experimental evidence suggesting that varenicline increases the risk of cardiovascular events. Varenicline progresses atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. This adverse effect is likely due to enhanced net uptake of oxidized low-density lipoprotein (oxLDL) in macrophages as a result of increased scavenger receptors and decreased cholesterol efflux transporters. However, a regimen has not yet been presented for avoidance or amelioration of the risk for varenicline-induced cardiovascular events. The aim of this study was to examine the effect of hesperidin, a citrus flavonoid, on varenicline-aggravated atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. Hesperidin inhibited the aggravating effect of varenicline in the whole aorta, aortic arch, and aortic root of ApoE KO mice. In addition, hesperidin protected against varenicline-enhanced oxLDL net uptake by blocking the increased expression of CD36 and LOX-1 scavenger receptors and decreased expression of ABCA1 and ABCG1 cholesterol efflux transporters in RAW 264.7 cells. Our findings suggest that hesperidin can avoid or ameliorate the risk for cardiovascular events induced by varenicline treatment.

A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1α.

BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that down-regulates hepatic low-density lipoprotein receptor (LDLR) by binding and shuttling LDLR to lysosomes for degradation. The development of therapy that inhibits PCSK9 has attracted considerable attention for the management of cardiovascular disease risk. However, only monoclonal antibodies of PCSK9 have reached the clinic use. Oral administration of small-molecule transcriptional inhibitors has the potential to become a therapeutic option.MethodsHere, we developed a cell-based small molecule screening platform to identify transcriptional inhibitors of PCSK9. Through high-throughput screening and a series of evaluation, we found several active compounds. After detailed investigation on the pharmacological effect and molecular mechanistic characterization, 7030B-C5 was identified as a potential small-molecule PCSK9 inhibitor.FindingsOur data showed that 7030B-C5 down-regulated PCSK9 expression and increased the total cellular LDLR protein and its mediated LDL-C uptake by HepG2 cells. In both C57BL/6 J and ApoE KO mice, oral administration of 7030B-C5 reduced hepatic and plasma PCSK9 level and increased hepatic LDLR expression. Most importantly, 7030B-C5 inhibited lesions in en face aortas and aortic root in ApoE KO mice with a slight amelioration of lipid profiles. We further provide evidences suggesting that transcriptional regulation of PCSK9 by 7030B-C5 mostly depend on the transcriptional factor HNF1α and FoxO3. Furthermore, FoxO1 was found to play an important role in 7030B-C5 mediated integration of hepatic glucose and lipid metabolism.Interpretation7030B-C5 with potential suppressive effect of PCSK9 expression may serve as a promising lead compound for drug development of cholesterol/glucose homeostasis and cardiovascular disease therapy.FundThis work was supported by grants from the National Natural Science Foundation of China (81473214, 81402929, and 81621064), the Drug Innovation Major Project of China (2018ZX09711001-003-006, 2018ZX09711001-007 and 2018ZX09735001-002), CAMS Innovation Fund for Medical Sciences (2016-I2M-2-002, 2016-I2M-1-011 and 2017-I2M-1-008), Beijing Natural Science Foundation (7162129).

Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet

Age-related hearing loss (ARHL) is a neurodegenerative disease associated with an aged population. ARHL is influenced by biological factors such as aging, sex difference, and atherosclerosis. The mechanisms of ARHL caused by atherosclerosis have not been previously determined in apolipoprotein E knockout (ApoE KO) male mice. To investigate the onset and cause of the hearing loss, ApoE KO male mice were treated with a western diet (ApoE KO-WD) for 16 weeks. The lipid profile, atherosclerotic plaques throughout the aorta, and auditory brainstem response (ABR) thresholds were measured in the ApoE KO-WD male mice. The expression of S100 calcium-binding protein B (S100B), a neuronal damage biomarker, was also observed. Reactive oxygen species (ROS) and apoptosis rates were detected in the cochlea of the ApoE KO male mice. Atherosclerotic plaques on the aorta and ABR thresholds were significantly increased in the ApoE KO-WD male mice at 24 weeks of age. ABR thresholds had a statistically significant positive correlation with the area of atherosclerotic plaques (r = 0.783, p = 0.013) in male mice at 24 weeks of age. S100B protein expression and the dihydroethidium (DHE) reaction to ROS in the cochlear spiral ganglion neurons (SGNs) were significantly increased in the ApoE KO and ApoE KO-WD male mice. Cells positive for active caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) in the SGNs were significantly increased in ApoE KO-WD male mice indicating an increased rate of cellular apoptosis. In conclusion, ROS in the SGNs were activated by increased S100B expression in ApoE KO-WD male mice, and this resulted in an increased apoptosis rate. Thus, hearing loss began at 16 weeks in ApoE KO-WD male mice. Our results suggest that the ApoE KO-WD male mice are a suitable animal model for studying ARHL associated with exacerbated atherosclerosis.

Inhibition of Smooth Muscle Cell Proliferation and Migration by a Talin Modulator Attenuates Neointimal Formation after Femoral Arterial Injury.

Background and ObjectivesVascular smooth muscle cell (SMC) proliferation and migration play a critical role in neointimal formation. Focal adhesion is involved in cell proliferation and migration, and talin is known to be a key regulator of these processes. We synthesized a new talin modulator that binds to the talin protein, and investigated its effects on SMCs and neointimal formation after vascular injury.MethodsHuman aortic SMCs (HAoSMCs) were treated with a newly synthesized talin modulator. Apolipoprotein E knockout (ApoE KO) mice were subjected to left femoral arterial injury and orally administered with the talin modulator daily. Laser Doppler imager was used to compare the blood flow, and injured femoral arteries and blood serum were analyzed after 28 days.ResultsThe talin modulator significantly inhibited cell proliferation in a concentration-dependent manner and suppressed the migration of HAoSMCs. Treatment with a talin modulator resulted in a significant reduction in the phosphorylation of focal adhesion molecules and downstream signaling molecules related to cell proliferation and migration. The effects of the talin modulator in HAoSMCs were found to be reversible, as evidenced by the reactivation of signaling pathways upon its removal. After 28 days of administration of the talin modulator, an improvement in the blood flow and reduction in neointimal formation in the injured femoral arteries were observed.ConclusionsWe demonstrated the inhibitory effects of a talin modulator on SMC proliferation and migration, and that were associated with downregulation of signaling pathways, resulting in the attenuation of neointimal formation in ApoE KO mice.

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.

Abstract 198: Metabolic Stress Causes Mitochondrial Dysfunction and Vascular Senescence via Angiotensin II Type I Receptor Signal / Rho Kinase Induced Mitochondrial Fission

Introduction: Metabolic stress, such as oxidized low density lipoprotein (ox-LDL) and advanced glycation end products (AGE) cause mitochondrial dysfunction and evoke vascular senescence and atherosclerosis. Mitochondria are highly dynamic organelles that constantly change their morphology to fusion or fission. This study aims to clarify whether mitochondrial dynamics is involved in the etiology of vascular senescence. Methods: We used C57BL6 (WT), apolipoprotein E deficient (ApoE KO) and db/db mice. We also conducted in vitro experiments using VSMC and HUVEC. Results: The degree of arterial senescence, arterial protein level of Drp1 in mitochondrial fraction and mitochondrial oxidative stress were higher and the number of fused mitochondria and mitochondrial function were lower in either ApoE KO or db/db mice than those of WT mice. Treatment with Drp1-specific inhibitor, mdivi-1, to these mice reduced excessive mitochondrial fission, oxidative stress and attenuated vascular senescence. Administration of either ox-LDL or AGE to cells also induced excessive mitochondrial fission though phosphorylation of Drp1 at Ser616, mitochondrial dysfunction, reactive oxygen species production and cellular senescence. Treatment with mdivi-1 to these cells restored imbalance of mitochondrial dynamics and these detrimental alterations. These results suggest that metabolic stress-induced mitochondrial dysfunction and cellular senescence were derived from Drp1-dependent mitochondrial fission. Treatment with angiotensin II type1 receptor (AT1R) blocker (ARB) to either Apo E KO mice or db/db in in vivo experiments and administration of ARB to cells with ox-LDL or AGE stimulation in in vitro experiments inactivated Drp1 and improved imbalance of mitochondrial dynamics, mitochondrial dysfunction and cellular senescence, suggesting that AT1R signal is involved in regulating metabolic stress-induced mitochondrial fission. Finally, inhibition of Rho kinase ROCK1 successfully attenuated Drp1-mediated mitochondrial fission and cellular senescence derived from metabolic stress. Conclusion: Metabolic stress causes cellular and vascular senescence through AT1R signal/Rho kinase-mediated mitochondrial fission.

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.

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.

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.

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 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 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.

Fimasartan reduces neointimal formation and inflammation after carotid arterial injury in apolipoprotein E knockout mice

BackgroundThe beneficial effects of angiotensin II type 1 receptor blockers (ARBs) on atherosclerosis have been demonstrated in numerous studies. We investigated the effects of fimasartan on reducing neointimal formation and systemic inflammation after carotid artery (CA) injury in Apolipoprotein E knockout (ApoE KO) mice.MethodsApoE KO mice were randomly allocated to Group I (without CA injury), Group II (without CA injury + Fimasartan), Group III (CA injury), and Group IV (CA injury + Fimasartan). Fimasartan was orally administered everyday starting 3 days before iatrogenic left CA injury.ResultsAt 28 days, neointimal hyperplasia and the inflammatory cytokines including TNFα, IL-6, ICAM, and MMP-9 in the peripheral blood were significantly reduced in Groups II and IV compared to Groups I and III, respectively.All fimasartan-administered groups revealed significant increases of CD4+CD25+Foxp3+ regulatory T (Treg) cells with increased plasma levels of IL-10 and TGFβ. In addition, increased CD8+ T cells by fimasartan were correlated with reduced smooth muscle cell (SMC) proliferation in the neointima in Groups II and IV. Furthermore, the populations of Treg and CD8+ T cells in total splenocytes were increased in Groups II and IV compared to Groups I and III, respectively. The enlargement of spleens due to CA injury in the Group III was attenuated by fimasartan, as shown in the Group IV. These data indicate that fimasartan significantly reduced SMC proliferation in neointima and increased Treg cells in ApoE KO CA injury mice.ConclusionsThis study suggests fimasartan could be an efficient strategy for reduction of atherosclerotic progression, with a decrease in immune response and systemic inflammation.

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.

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.

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 & Co., Inc., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi.