Atherosclerosis In apoE-KO Mice Research Articles

Protective effects of imeglimin on the development of atherosclerosis in ApoE KO mice treated with STZ

BackgroundImeglimin is a new anti-diabetic drug which promotes insulin secretion from pancreatic β-cells and reduces insulin resistance in insulin target tissues. However, there have been no reports examining the possible anti-atherosclerotic effects of imeglimin. In this study, we investigated the possible anti-atherosclerotic effects of imeglimin using atherosclerosis model ApoE KO mice treated with streptozotocin (STZ).MethodsApoE KO mice were divided into three groups: the first group was a normoglycemic group without injecting STZ (non-DM group, n = 10). In the second group, mice were injected with STZ and treated with 0.5% carboxymethyl cellulose (CMC) (control group, n = 12). In the third group, mice were injected with STZ and treated with imeglimin (200 mg/kg, twice daily oral gavage, n = 12). We observed the mice in the three groups from 10 to 18 weeks of age. Plaque formation in aortic arch and expression levels of various vascular factors in abdominal aorta were evaluated for each group.ResultsImeglimin showed favorable effects on the development of plaque formation in the aortic arch in STZ-induced hyperglycemic ApoE KO mice which was independent of glycemic and lipid control. Migration and proliferation of vascular smooth muscle cells and infiltration of macrophage were observed in atherosclerotic lesions in STZ-induced hyperglycemic ApoE KO mice, however, which were markedly reduced by imeglimin treatment. In addition, imeglimin reduced oxidative stress, inflammation and inflammasome in hyperglycemic ApoE KO mice. Expression levels of macrophage makers were also significantly reduced by imeglimin treatment.ConclusionsImeglimin exerts favorable effects on the development of plaque formation and progression of atherosclerosis.

TNF-α Preconditioning Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in an Experimental Model of Atherosclerosis.

Atherosclerosis (AS) is an inflammatory disease involving multiple factors in its initiation and development. In recent years, the potential application of mesenchymal stem cells (MSCs) for treating AS has been investigated. This study examined the effect of TNF-α preconditioning on MSCs' therapeutic efficacy in treating AS in ApoE KO mice. TNF-α-treated MSCs were administered to high-fat diet-treated ApoE KO mice. Cytokine and serum lipid levels were measured before and after treatment. Cryosections of the atherosclerotic aorta were stained with Oil-Red-O, and the relative areas of atherosclerotic lesions were measured. The level of Tregs were increased in TNF-α-MSC-treated animals compared to the MSCs group. In addition, the systemic administration of TNF-α-MSCs to ApoE KO mice reduced the level of proinflammatory cytokines such as TNF-α and IFN-γ and increased the level of the immunosuppressive IL-10 in the blood serum. Total cholesterol and LDL levels were decreased, and HDL levels were increased in the TNF-α-MSCs group of ApoE KO mice. A histological analysis showed that TNF-α-MSCs decreased the size of the atherosclerotic lesion in the aorta of ApoE KO mice by 38%, although there was no significant difference when compared with untreated MSCs. Thus, our data demonstrate that TNF-α-MSCs are more effective at treating AS than untreated MSCs.

462-P: DPP-4 Deficiency Suppresses the Progression of Atherosclerosis via Inducing M2 Macrophage Polarization

Background: Dipeptidyl peptidase-4 (DPP4) inhibitors suppressed progression of atherosclerosis in atherosclerotic mouse models. However, the role of DPP4 on the progression of atherosclerosis is not clear. We investigated the effect of DPP4 deficiency on the progression of atherosclerosis in DPP4 and apolipoprotein-E double knockout (DPP4/ApoE-DKO) mice. Methods: Both ApoE-KO mice and DPP4/ApoE-DKO mice were fed with normal chow until 20 weeks of age. Whole aorta and aortic sinus were stained with Oil red O. Bone marrow-derived macrophages (mφ) from wildtype or DPP4-KO mice were used in in vitro study. Expression of iNOS, MCP-1, TNF-α and IL-1β (M1 markers), and arginase-1, Mgl2 and Ym1 (M2 markers) mRNA were evaluated by real-time RT-PCR, and mφ polarization was evaluated by flowcytometry. Results: There were no differences on food intake, body weight, lipid profiles and the blood glucose levels by insulin tolerance test between each group, but the blood glucose levels by glucose tolerance test were significantly decreased in DPP4/ApoE-DKO mice. Oil-Red-O staining demonstrated that the size of atherosclerotic lesions was significantly smaller in DPP4/ApoE-DKO mice. In in vitro assay, LPS+IFNγ induced the mRNA expression of M1 markers and there was no difference between wildtype mφ and DPP4-KO mφ. On the other hand, the mRNA expression of M2 markers were significantly increased in DPP4-KO mφ. Flowcytometry revealed that the number of iNOS (M1 marker)-positive cells were increased by LPS+IFNγ, which was comparable between wildtype mφ and DPP4-KO mφ. On the other hand, the number of CD206 (M2 marker)-positive cells was higher in DPP4-KO mφ than that in wildtype mφ. Conclusion: This study suggests that DPP4 deficiency suppresses the progression of atherosclerosis in apoE-KO mice. Moreover, DPP4 deficiency increases the polarization of M2 mφ. These findings may indicate the involvement of DPP4 at least in part on the development of diabetic macrovascular complications. Disclosure Y.Zhou: None. T.Matsumura: Research Support; Shimadzu Corporation, Speaker's Bureau; Eli Lilly Japan K.K., Boehringer Ingelheim Japan, Inc. Y.Yagi: None. T.Yoshinaga: None. T.Senokuchi: None. N.Ishii: None. T.Kondo: None. E.Araki: Research Support; Eli Lilly Japan K.K., Nippon Boehringer Ingelheim, Mitsubishi Tanabe Pharma Corporation, Speaker's Bureau; AstraZeneca, Merck & Co., Inc., Ono Pharmaceutical Co., Ltd., Kowa Company, Ltd., Sumitomo Pharma Co., Ltd., Daiichi Sankyo, Novo Nordisk Pharma Ltd.

Dietary choline supplementation increases the plasma concentration of several amino acids during atherosclerosis development

Background and Aims : Increased dietary intake of choline worsens the development of atherosclerosis in ApoE-KO mice. Gut microbial metabolism of choline results in the production of trimethylamine, which, upon intestinal absorption is converted into the pro-atherogenic molecule trimethylamine-N-oxide (TMAO) in the liver. In this study we investigated if additional endogenous plasma metabolites were modulated by increased choline intake.

The sodium‐glucose co‐transporter 2 inhibitor tofogliflozin suppresses atherosclerosis through glucose lowering in ApoE‐deficient mice with streptozotocin‐induced diabetes

Epidemiological and animal studies have revealed that sodium‐glucose cotransporter 2 (SGLT2) inhibitors suppress cardiovascular events in subjects with type 2 diabetes and atherosclerosis in animal models of diabetes. However, it still remains unclear if the anti‐atherosclerotic effect of SGLT2 inhibitors is entirely dependent on their glucose‐lowering effect. Tofogliflozin, a highly specific SGLT2 inhibitor, was administrated to apolipoprotein‐E‐deficient (ApoEKO) with streptozotocin (STZ)‐induced diabetes and nondiabetic ApoEKO mice. After 6 weeks, samples were collected to investigate the histological changes and peritoneal macrophage inflammatory cytokine levels. Tofogliflozin suppressed atherosclerosis in the diabetic ApoEKO mice. The atherosclerosis lesion areas and accumulation of macrophages in these areas were reduced by tofogliflozin treatment. The expression levels of interleukin (IL)‐1β and IL‐6 in the peritoneal macrophages were significantly suppressed in the tofogliflozin‐treated diabetic ApoEKO mice. Tofogliflozin treatment failed to inhibit atherosclerosis in the nondiabetic ApoEKO mice. No significant difference in the anti‐atherosclerotic effects of insulin and tofogliflozin was observed between diabetic ApoEKO mice with equivalent degrees of glycemic control achieved with the two treatments. Insulin treatment significantly reduced the IL‐1β and IL‐6 expression levels in the peritoneal macrophages of the diabetic ApoEKO mice. Significant decrease of the LPS‐stimulated IL‐1β concentrations was also observed in the conditioned medium of the peritoneal macrophages collected from insulin‐ and tofogliflozin‐treated diabetic ApoEKO mice. These results suggest that tofogliflozin suppresses atherosclerosis by improving glucose intolerance associated with inhibition of inflammation. Tofogliflozin suppresses atherosclerosis in ApoEKO mice with STZ‐induced diabetes via its glucose‐lowering effect.

20(S)-Protopanaxadiol decreases atherosclerosis in ApoE KO mice by increasing the levels of LDLR and inhibiting its binding with PCSK9.

Chinese medicinal and edible plants such as Panax notoginseng and ginseng are widely used for the treatment of atherosclerosis (AS). AS is the main pathological basis of cardiac-cerebral vascular disease, which seriously threatens human health and quality of life. Low-density lipoprotein (LDL) is the main pathogenic factor of AS. The LDL receptor (LDLR) is an important protein that functions to mediate the uptake and degradation of plasma LDL. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) can mediate the internalization and degradation of LDLR. So, increasing the LDLR level by inhibiting PCSK9 is an important means of prevention and treatment of AS. In this study, by combining interaction technology (surface plasmon resonance, SPR) of small molecule compounds with membrane receptor proteins, cell experiments, and in vivo experiments, it is proved for the first time that 20(S)-protopanaxadiol (PPD), as a hydrolytic product of Panax notoginseng saponins in the intestinal tract, can bind to the extracellular domain of LDLR and inhibit the role of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mediating LDLR degradation. The results showed that PPD significantly reduced aortic plaques and hepatic steatosis in HFD-fed ApoE KO mice. LDLR protein levels were elevated in the liver tissues isolated from PPD-treated HFD-fed ApoE KO mice and PPD-treated HepG2 cells. Our findings demonstrated that PPD significantly increased LDLR levels and reduced AS in the HFD-fed ApoE KO mice on account of LDLR degradation being inhibited by PPD inhibiting the interaction between PCSK9 and LDLR.

Abstract 13311: A Novel Senolytic Drug, Seno-7284 Ameliorates Age-Related Cardiometabolic Diseases

Cellular senescence plays a pivotal role in aging and age-related disorders. Recently, it was shown that elimination of senescent cells, so-called "senolysis" has the potential to become a next generation therapy for age-related disorders including cardio-metabolic diseases. However, currently there is no senolytic drug available in clinical settings. The present study was aimed to identify a novel senolytic reagent effective for cardiometabolic diseases in compounds already available in clinical settings. We demonstrate a compound called "Seno-7284" exhibits senolytic effect in murine models of type 2 diabetes, atherosclerosis, progeroid and chronological aged mice. Seno-7284 reduced the accumulation of senescent cells in visceral adipose tissue of diabetic mice as senescence-associated-beta-galactosidase (SA-beta-gal) staining exhibits. This associated with the suppression in systemic glucose intolerance, and adipose tissue inflammation in four weeks after the administration of Seno-7284. Administrating Seno-7284 for two weeks also suppressed the progression of atherosclerosis in ApoE-KO mice. This drug significantly improved the lifespan of Zm-KO mice by administering it from 12 weeks old. Administration of Seno-7284 from 50 week-old for 20 weeks improves physical function in chronological aged mice. Further analysis including single cell RNA-seq suggested that Seno-7284 stimulates endogenous senolytic function of NK cells and CD8+ T cells. Our results indicate Seno-7284 mediates its biological effects by inducing senolysis in murine aging models. Seno-7284 would become promising therapies for age-related disorders.

Deficiency of LMP10 Attenuates Diet-Induced Atherosclerosis by Inhibiting Macrophage Polarization and Inflammation in Apolipoprotein E Deficient Mice.

Macrophage polarization and inflammation are key factors for the onset and progression of atherosclerosis. The immunoproteasome complex consists of three inducible catalytic subunits (LMP2, LMP10, and LMP7) that play a critical role in the regulation of these risk factors. We recently demonstrated that the LMP7 subunit promotes diet-induced atherosclerosis via inhibition of MERTK-mediated efferocytosis. Here, we explored the role of another subunit of LMP10 in the disease process, using ApoE knockout (ko) mice fed on an atherogenic diet (ATD) containing 0.5% cholesterol and 20% fat for 8 weeks as an in vivo atherosclerosis model. We observed that ATD significantly upregulated LMP10 expression in aortic lesions, which were primarily co-localized with plaque macrophages. Conversely, deletion of LMP10 markedly attenuated atherosclerotic lesion area, CD68+ macrophage accumulation, and necrotic core expansion in the plaques, but did not change plasma metabolic parameters, lesional SM22α+ smooth muscle cells, or collagen content. Myeloid-specific deletion of LMP10 by bone marrow transplantation resulted in similar phenotypes. Furthermore, deletion of LMP10 remarkably reduced aortic macrophage infiltration and increased M2/M1 ratio, accompanied by decreased expression of pro-inflammatory M1 cytokines (MCP-1, IL-1, and IL-6) and increased expression of anti-inflammatory M2 cytokines (IL-4 and IL-10). In addition, we confirmed in cultured macrophages that LMP10 deletion blunted macrophage polarization and inflammation during ox-LDL-induced foam cell formation in vitro, which was associated with decreased IκBα degradation and NF-κB activation. Our results show that the immunoproteasome subunit LMP10 promoted diet-induced atherosclerosis in ApoE ko mice possibly through regulation of NF-κB-mediated macrophage polarization and inflammation. Targeting LMP10 may represent a new therapeutic approach for atherosclerosis.

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.

MicroRNA-377 Inhibits Atherosclerosis by Regulating Triglyceride Metabolism Through the DNA Methyltransferase 1 in Apolipoprotein E-Knockout Mice.

Lipoprotein lipase (LPL) plays an important role in triglyceride metabolism. It is translocated across endothelial cells to reach the luminal surface of capillaries by glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), where it hydrolyzes triglycerides in lipoproteins. MicroRNA 377 (miR-377) is highly associated with lipid levels. However, how miR-377 regulates triglyceride metabolism and whether it is involved in the development of atherosclerosis remain largely unexplored. Methods and Results: The clinical examination displayed that miR-377 expression was markedly lower in plasma from patients with hypertriglyceridemia compared with non-hypertriglyceridemic subjects. Bioinformatics analyses and a luciferase reporter assay showed that DNA methyltransferase 1 (DNMT1) was a target gene of miR-377. Moreover, miR-377 increased LPL binding to GPIHBP1 by directly targeting DNMT1 in human umbilical vein endothelial cells (HUVECs) and apolipoprotein E (ApoE)-knockout (KO) mice aorta endothelial cells (MAECs). In vivo, hematoxylin-eosin (H&E), Oil Red O and Masson's trichrome staining showed that ApoE-KO mice treated with miR-377 developed less atherosclerotic plaques, accompanied by reduced plasma triglyceride levels. It is concluded that miR-377 upregulates GPIHBP1 expression, increases the LPL binding to GPIHBP1, and reduces plasma triglyceride levels, likely through targeting DNMT1, inhibiting atherosclerosis in ApoE-KO mice.

Abstract 211: MKL1 Affects Proliferation/Survival Property of Pro-Atherogenic Macrophages

Coronary artery disease (CAD) is one of the leading causes of death in the most countries. Atherosclerosis of coronary arteries often results in high incidence of vascular occlusion and is recognized as the major cause of CAD. We have previously reported from a genome-wide association study that a promoter SNP in megakaryoblastic leukemia 1 gene ( MKL1 ), which conferred higher transcriptional activity, was significantly associated with the susceptibility to CAD as well as coronary atherosclerosis. MKL1 is a Rho-Rock signaling-responsive co-activator of serum response factor, and regulates a variety of cellular functions. We found that MKL1 was highly expressed in activated macrophages in the neointima of atherosclerotic lesions from human and ApoE-knockout (KO) mouse. In addition, the expression level of MKL1 in lesional macrophages was increased during the development of atherosclerosis in ApoE-KO mice. We have established a transgenic mouse line, MKL1-TgM, in which human MKL1 was specifically overexpressed in monocyte/macrophage lineage cells. In this study, MKL1-TgM was crossbred onto ApoE-KO background. ApoE-KO/MKL1-TgM fed with normal chow developed severer atherosclerosis and showed poorer survival rate than ApoE-KO mice. Serum level of pro-inflammatory cytokines was increased in ApoE-KO/MKL1-TgM. Interestingly, ApoE-KO/MKL1-TgM also developed hypertriglyceridemia, which was associated with severe lipodystrophy. We also investigated how MKL1 regulated macrophages function. Recently, local proliferation/accumulation of lesional macrophages was reported to be a crucial event in atherosclerosis. We treated bone marrow-derived macrophages (BMDMs) with stimuli, such as M-CSF or OxLDL, and found that proliferation property of BMDMs from MKL1-TgM was elevated. On the other hand, BMDMs from MKL1-TgM were more resistant to apoptosis. Furthermore, immunohistochemistry analyses of lesional macrophages in ApoE-KO/MKL1-TgM showed enhanced proliferation and mitigated apoptosis. Taken together, our data indicated that MKL1 would contribute to the development of atherosclerosis by modulating pro-atherogenic functions of macrophages, providing a potential molecular target for the therapy and prevention of atherosclerosis.

Angiotensin-(1-7)-induced Mas receptor activation attenuates atherosclerosis through a nitric oxide-dependent mechanism in apolipoproteinE-KO mice

Angiotensin (Ang)-(1-7) ameliorates vascular injury by increasing nitric oxide (NO) bioavailability. Evidence that Ang-(1-7) attenuates the development of atherosclerosis through a NO-dependent mechanism is still missing. Moreover, it has been postulated that Ang-(1-7) may mediate its effects by other mechanisms than Mas receptor activation. To investigate Ang-(1-7)-dependent Mas receptor function, we treated apoE-KO and apoE/Mas-KO mice chronically with Ang-(1-7) (82μg/kg per hour) or saline for 6weeks. Flow-mediated dilation (FMD), a measure for NO-dependent vasodilation and the most accepted prognostic marker for the development of atherosclerosis, was measured in vivo. Chronic Ang-(1-7) treatment improved FMD and attenuated the development of atherosclerosis in apolipoproteinE (apoE)-KO but not in apoE/Mas-KO mice. These effects were accompanied by increased aortic nitrite and cGMP levels. To test whether Ang-(1-7) modulates atherosclerosis through a NO-dependent mechanism, apoE-KO mice were treated with the NO synthase inhibitor L-NAME (20mg/kg/day) in the presence or absence of Ang-(1-7). L-NAME treatment reduced aortic nitrite content and increased blood pressure and exaggerated atherosclerosis compared to untreated apoE-KO mice. In L-NAME-treated apoE-KO mice, chronic Ang-(1-7) treatment did not increase aortic nitrite content and consequently showed no effect on blood pressure and the development of atherosclerosis. The present study proves that Ang-(1-7) mediates its protective vascular effects through Mas receptor activation. Moreover, Ang-(1-7)-mediated NO generation is essential for improving vascular function and prevents atherosclerosis in apoE-KO mice.

Procyanidins extracted from the litchi pericarp attenuate atherosclerosis and hyperlipidemia associated with consumption of a high fat diet in apolipoprotein-E knockout mice

The beneficial effects of red wine against cardiovascular disease are associated with the abundant antioxidant polyphenols such as procyanidins. Recently, procyanidins extracted from the litchi pericarp (LPPC), a new source of procyanidins showed strong antioxidant activities in vitro, have been isolated and identified in our laboratory. The aim of present study was to investigate the anti-atherosclerotic effects of LPPC on atherosclerosis and hyperlipidemia in apolipoprotein E knockout (ApoE KO) mice fed a high fat diet (HFD, 21% fat, 0.15% cholesterol) for 24 weeks. The results showed that LPPC intervention alleviated atherosclerosis, fat accumulation and hyperlipidemia in ApoE KO mice. Furthermore, real-time RT-PCR results showed that LPPC can regulate several key genes involved in hepatic lipid homeostasis, such as increasing mRNA levels of farnesoid X receptor (FXR) and small heterodimer partner (SHP) which emerge as key regulators of lipid homeostasis at the transcriptional level, decreasing mRNA levels of 3-hydroxy-3-Methylglutaryl (HMG)-CoA reductase which mediates cholestrol biosynthesis, and up-regulating the mRNA expressions of ATP-binding cassette transporter-1 (ABCA1) which modulates cholesterol efflux. Thus, these results elucidated that LPPC could alleviate the lipid disorder especially hypercholesteromia and ameliorate atherosclerosis in ApoE-KO mice fed a WTD via regulating gene expression involved in hepatic lipid homeostasis effectively.

Procyanidins extracted from the litchi pericarp ameliorate atherosclerosis in ApoE knockout mice: their effects on nitric oxide bioavailability and oxidative stress.

Epidemiological studies strongly support the role of procyanidin-rich beverages and fruit in the prevention of cardiovascular diseases. Procyanidins extracted from the litchi pericarp (LPPC), a new source of procyanidins, were isolated and identified in our laboratory and have been proven to possess strong antioxidant activity in vitro. In the present study, we investigated the anti-atherosclerotic effects of LPPC in apolipoprotein E knockout (ApoE KO) mice fed a high fat diet for 24 weeks. LPPC (100 mg kg-1 body mass daily) significantly reduced the atherosclerotic lesion size (P < 0.01 versus ApoE KO mice), excess NO production and iNOS expression. Moreover, the mRNA and protein expressions of eNOS in the aortas of ApoE KO mice were increased by LPPC which further explains the endothelial protective action of LPPC. LPPC also showed profound antioxidant effects in ApoE KO mice. Plasma TBARS contents and the mRNA expression for NADPH oxidase (p47phox, p67phox, NOX-2/gp91phox and NOX-4) in the aortas of ApoE KO mice were significantly reduced, whereas plasma SOD activity was markedly elevated by LPPC. These results elucidated that LPPC could ameliorate atherosclerosis in ApoE-KO mice by improving NO bioavailability and reducing oxidative stress through NADPH oxidase-dependent mechanisms.

Protective effect of betulinic acid on early atherosclerosis in diabetic apolipoprotein-E gene knockout mice

Atherosclerosis, a chronic and progressive disease, is a leading cause of endothelial dysfunction, diabetes mellitus, hypertension, and hypercholesterolemia. Betulinic acid (BA), a pentacyclic triterpene, has been reported to have a variety of biological effects, including anti-inflammatory and immunomodulatory properties. This study was designed to determine whether BA could prevent atherosclerosis in diabetic apolipoprotein-E gene knockout (ApoE KO) mice. The mice were treated with BA for 12 weeks to examine its beneficial effects on atherosclerosis in ApoE KO mice. Male ApoE KO mice and age-matched control group mice (C57BL/6Jms) were used as experimental systems and their systolic blood pressure, insulin resistance, and vascular inflammation were measured. BA-treated ApoE KO mice showed lowered systolic blood pressure. The metabolic parameter showed that BA decreased blood urea nitrogen, triglyceride, and total cholesterol levels. Blood glucose, insulin, glucose tolerance results, and the homeostasis model assessment of insulin resistance (HOMA-IR) index were found to be better in BA-treated ApoE KO mice than untreated ApoE KO mice. Consistent with the change in lipid profiles, oil red O and H&E staining revealed that treatment with BA reduced atherosclerotic lesions such as roughened endothelial layers. BA ameliorated the reduction of endothelial nitric oxide synthase (eNOS) expression, leading to the inhibition of intracellular adhesion molecule 1 (ICAM-1) and endothelin 1 (ET-1) expression. These results suggest that BA may be useful in the treatment and prevention of early atherosclerosis via the attenuation of endothelial dysfunction in diabetic ApoE KO mice.

Long-chain monounsaturated fatty acid-rich fish oil attenuates the development of atherosclerosis in mouse models.

Fish oil-derived long-chain monounsaturated fatty acids (LCMUFA) containing chain lengths longer than 18 were previously shown to improve cardiovascular disease risk factors in mice. However, it is not known if LCMUFA also exerts anti-atherogenic effects. The main objective of the present study was to investigate the effect of LCMUFA on the development of atherosclerosis in mouse models. LDLR-KO mice were fed Western diet supplemented with 2% (w/w) of either LCMUFA concentrate, olive oil, or not (control) for 12 wk. LCMUFA, but not olive oil, significantly suppressed the development of atherosclerotic lesions and several plasma inflammatory cytokine levels, although there were no major differences in plasma lipids between the three groups. At higher doses 5% (w/w) LCMUFA supplementation was observed to reduce pro-atherogenic plasma lipoproteins and to also reduce atherosclerosis in ApoE-KO mice fed a Western diet. RNA sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated PPAR signaling pathways in liver. In cell culture studies, apoB-depleted plasma from LDLR-K mice fed LCMUFA showed greater cholesterol efflux from macrophage-like THP-1 cells and ABCA1-overexpressing BHK cells. Our research showed for the first time that LCMUFA consumption protects against diet-induced atherosclerosis, possibly by upregulating the PPAR signaling pathway.

P110Delta Inhibits Monocyte Infiltration by Thioglycollate-Induced Periotoneal Inflammation but Not HCD-Induced Inflammation and Atherosclerosis in APOE KO Mice.

We have previously reported that phosphoinositide 3-kinase p110δ knockout (p110δ KO) diminished the adhesion of leukocytes to capillary venules and suppressed the peritoneal infiltration of leukocytes, both functions that play important roles in atherosclerosis. Therefore, we hypothesized that p110δ deficiency might be protective against atherosclerosis. Apolipoprotein E knockout (ApoE KO) mice were crossed with p110δ KO mice to generate homozygous double knockout mice (ApoE/p110δ DKO). The present study showed that ApoE/p110δ DKO mice fed with a high cholesterol diet (HCD) demonstrated less peritoneal infiltration of leukocytes and monocytes compared with ApoE KO mice after intraperitoneal injection of thioglycollate, an inducer of acute peritoneal inflammation. Unexpectedly, atherosclerosis in the aortic root and in the entire aorta was similar between the ApoE/p110δ DKO and ApoE KO groups. No difference in Mac-3 expression, indicative of macrophage infiltration, was found between the two groups. Further analysis showed that ApoE KO mice chronically fed with HCD had increased levels of total cholesterol, low-density lipoprotein in the blood and counts and percentages of circulating monocytes compared with ApoE KO mice fed with a normal diet. Consistently, the deficiency of p110δ affected neither the counts nor the percentages of monocytes nor the lipid profiles in the blood. The results suggested that p110δ plays an important role in acute but not in chronic inflammation, the latter being included in the early characteristics of atherosclerosis, which might explain the finding that p110δ deficiency fails to inhibit early atherosclerosis.

Abstract 28: Mechanistic Studies of HDL Mimetic Peptide ATI-5261 Reveal Aspects of Class A Alpha-helix Structure that Induce Cytotoxicity and Hypertriglyceridemia in vivo: Design of Safe Analogs with Potent Anti-atherosclerosis and Anti-diabetic Actions

HDL mimetic peptides hold promise for the treatment of cardio-metabolic diseases. Transient increases in plasma TG and/or safety concerns may limit their therapeutic applications however, particularly in high risk patients with hypertriglyceridemia and diabetes. Presently, we sought to identify peptide structural features responsible for these adverse effects. The single helix peptide ATI-5261 was used as a model, since it forms a class A α-helix like many HDL mimetic peptides and has exceptional secondary structure and solubility. High dose ip injection (300 mg/kg) of ATI-5261 induced muscle toxicity in C57Bl/6 mice vs. vehicle (serum CPK@4 h = 32,314±2359 vs 143±44 IU/L), increased ALT and AST activities (233±10 &amp; 4595±933 vs 26±6 &amp; 84±21 IU/L) and elevated plasma TG (1951±77 vs 37±7 mg/dl). A majority (~90%) of the cytotoxicity and TG increase was eliminated by removing phenylalanine residues from the apolar face of ATI-5261. Similar results were obtained by removing cationic arginine residues from the lipid-water interface. Thus both features were necessary, but each not sufficient, to induce cytotoxicity and TG elevations. Based on this, a peptide (CS6253) was created that was non-toxic (no adverse effect level &gt;500 mg/kg; t1/2 =7.0±0.6 h in rats), retained secondary structure (75±5% α-helicity), high solubility and ability to stimulate ABCA1-dependent cholesterol efflux efficiently (Km=0.80±0.40 vs. 0.86±0.25 μg/ml ATI-5261). In vivo CS6253 was TG neutral, promoted macrophage RCT, and reduced (32%) substantial atherosclerosis in apoE KO mice fed western diet 10 wks (lesion area = 22±4 vs. 15±2% with 30 mg CS6253/kg, ip injection every 48 h for 6 wks, p&lt;0.01) and improved glucose - and insulin- tolerance tests with lowering of HbA1c levels in C57Bl/6 mice fed high-fat diet (DIO model). In summary, the cytotoxic and TG elevating effects of high-dose ATI-5261 were dependent on class A α-helix structure. CS6253 with its improved safety margin represents a novel ABCA1 ligand peptide with potential to treat diabetes and associated cardiovascular disease.

RNA interference of myocyte enhancer factor 2A accelerates atherosclerosis in apolipoprotein E-deficient mice.

ObjectiveMyocyte enhancer factor-2A (MEF 2A) has been shown to be involved in atherosclerotic lesion development, but its role in preexisting lesions is still unclear. In the present study we aim to assess the role of MEF 2A in the progression of pre-existing atherosclerosis.MethodsEighty apolipoprotein E-deficient mice (APOE KO) were randomly allocated to control, scramble and MEF 2A RNA interference (RNAi) groups, and constrictive collars were used to induce plaque formation. Six weeks after surgery, lentiviral shRNA construct was used to silence the expression of MEF 2A. Carotid plaques were harvested for analysis 4 weeks after viral vector transduction. Inflammatory gene expression in the plasma and carotid plaques was determined by using ELISAs and real-time RT-PCR.ResultsThe expression level of MEF 2A was significantly reduced in plasma and plaque in the RNAi group, compared to the control and NC groups, whereas the expression level of pro-inflammatory cytokines was markedly increased. Silencing MEF 2A using lentiviral shRNA significantly reduced the plaque collagen content and fibrous cap thickness, as well as increased plaque area. However, silencing MEF 2A had no obvious effect on plaque lipid content.ConclusionsLentivirus-mediated MEF 2A shRNA accelerates inflammation and atherosclerosis in APOE KO mice, but has no effect on lipoprotein levels in plasma.

Liver-specific deletion of lipid phosphate phosphatase 3 exacerbates atherosclerosis in apoE-KO mice

Liver-specific deletion of lipid phosphate phosphatase 3 exacerbates atherosclerosis in apoE-KO mice