Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice.
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ABSTRACT: Recent evidence suggests that the essential amino acid leucine may be involved in systemic cholesterol metabolism. In this study, we investigated the effects of leucine supplementation on the development of atherosclerosis in apoE null mice.ApoE null mice were fed with chow supplemented with leucine (1.5% w/v) in drinking water for 8 week. Aortic atherosclerotic lesions were examined using Oil Red O staining. Plasma lipoprotein-cholesterol levels were measured with fast protein liquid chromatography. Hepatic gene expression was detected using real-time PCR and Western blot analyses.Leucine supplementation resulted in 57.6% reduction of aortic atherosclerotic lesion area in apoE null mice, accompanied by 41.2% decrease of serum LDL-C levels and 40.2% increase of serum HDL-C levels. The body weight, food intake and blood glucose level were not affected by leucine supplementation. Furthermore, leucine supplementation increased the expression of Abcg5 and Abcg8 (that were involved in hepatic cholesterol efflux) by 1.28- and 0.86-fold, respectively, and significantly increased their protein levels. Leucine supplementation also increased the expression of Srebf1, Scd1 and Pgc1b (that were involved in hepatic triglyceride metabolism) by 3.73-, 1.35- and 1.71-fold, respectively. Consequently, leucine supplementation resulted in 51.77% reduction of liver cholesterol content and 2.2-fold increase of liver triglyceride content. Additionally, leucine supplementation did not affect the serum levels of IL-6, IFN-γ, TNF-α, IL-10 and IL-12, but markedly decreased the serum level of MCP-1.Leucine supplementation effectively attenuates atherosclerosis in apoE null mice by improving the plasma lipid profile and reducing systemic inflammation.
Project description:Dystrophin of the dystrophin-glycoprotein complex connects the actin cytoskeleton to basement membranes and loss of dystrophin results in Duchenne muscular dystrophy. We have previously shown injury-induced neointima formation of the carotid artery in mice with the mdx mutation (causing dystrophin deficiency) to be increased. To investigate the role of dystrophin in intimal recruitment of smooth muscle cells (SMCs) that maintains plaque stability in atherosclerosis we applied a shear stress-modifying cast around the carotid artery of apolipoprotein E (ApoE)-null mice with and without the mdx mutation. The cast induces formation of atherosclerotic plaques of inflammatory and SMC-rich/fibrous phenotypes in regions of low and oscillatory shear stress, respectively. Unexpectedly, presence of the mdx mutation markedly reduced the development of the inflammatory low shear stress plaques. Further characterization of the low shear stress plaques in ApoE-null mdx mice demonstrated reduced infiltration of CD3(+) T cells, less laminin and a higher SMC content. ApoE-null mdx mice were also found to have a reduced fraction of CD3(+) T cells in the spleen and lower levels of cytokines and monocytes in the circulation. The present study is the first to demonstrate a role for dystrophin in atherosclerosis and unexpectedly shows that this primarily involves immune cells.
Project description:Current evidence suggests that intrauterine bisphenol A (BPA) exposure increases the risk of developing cardiovascular diseases in later stages of life. The beneficial effect of resveratrol (Rsv) on developmental programming of atherosclerosis lesions formation in offspring is seldom reported. Hence, we sought to study the effect of maternal Rsv in ameliorating perinatal BPA exposure-induced atherosclerosis lesions formation in adult offspring using the apolipoprotein E-deficient (ApoE-/-) mice model. The pregnant ApoE-/- mice were allocated into three groups: control, BPA, BPA + resveratrol (BPA + Rsv). The BPA group mice received BPA in their drinking water (1 μg/ml). BPA + Rsv group mice received BPA in their drinking water (1 μg/ml) and were treated orally with Rsv (20 mg kg-1 day-1). All the treatments were continued throughout the gestation and lactation period. Quantitative analysis of Sudan IV-stained aorta revealed a significantly increased area of atherosclerotic lesions in both female (p < 0.01) and male adult offspring mice (p < 0.01) in the BPA group. Supplementation with Rsv significantly reduced the BPA-induced atherosclerotic lesion development in the female offspring mice (p < 0.05). Transmission electron microscopy revealed the presence of a significantly high incidence of autophagic endothelial, smooth muscle, and macrophage cells in the aorta of BPA-exposed mice. Rsv treatment reduced the incidence of autophagic cells in BPA-exposed mice. In conclusion, maternal Rsv supplementation significantly prevents the BPA-induced atherosclerotic lesions formation in a sex-dependent manner potentially by acting as an autophagy modulator.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-021-03078-y.
Project description:Branched-chain amino acids (BCAA), Leu, and the signaling pathways they regulate have been reported to either improve or worsen adiposity and insulin sensitivity. Therefore, it is unclear whether dietary supplementation of Leu would be beneficial. To help address this question, we examined the effect of adding Leu (150 mmol/L; Expt. 1 and Expt. 2) or BCAA (109 mmol/L of each; Expt. 3) to the drinking water on diet-induced obesity (induced with a 60-kJ% fat diet) in singly housed C57BL6/J male mice for at least 14 wk. Liquid and solid food intakes were evaluated weekly along with body weight. During the last few weeks, several blood samples were taken at different times for plasma glucose, total cholesterol, or Leu measurements. Metabolic rate by indirect calorimetry, locomotor activity by light beam breaking, body composition by H1-NMR, and insulin tolerance were also determined. Compared with control, supplementation did not affect body weight, food intake, oxygen consumption, locomotor activity, body composition, insulin tolerance, or total cholesterol. In fed mice, this method of Leu supplementation only increased plasma Leu by 76% when the supplemented group was compared with control. On the other hand, after overnight food deprivation, the plasma Leu did not differ between these 2 groups, even though the mice in the supplemented group had continuous access to Leu-containing water during the solid food deprivation. Taken together, the results do not provide evidence that either Leu or BCAA supplementation of drinking water ameliorates diet-induced obesity in mice, although it may improve glycemia.
Project description:AimsAtherosclerosis is the primary cause of cardiovascular diseases and stroke. The current study evaluated the interventional effects of a naturally occurring compound Notoginsenoside R1 (NR1) on atherosclerosis in ApoE-/- mice.Methods and resultsThe atherosclerotic lesion was significantly alleviated by NR1 treatment and this attenuation was marked by reduction in lipid deposition, fibrosis and oxidative stress. Increased serum levels of GSH and SOD and decreased level of MDH were observed in NR1-treated ApoE-/- mice. NR1 treatment also significantly decreased the levels of CHO, TG, ox-LDL and increased the level of HDL. Additionally, the levels of inflammatory cytokines including IL-2, IL-6, TNF-α and γ-IFN were markedly reduced in NR1-treated ApoE-/- mice. Furthermore, significantly increased aortic expression of miR-26a, miR-21, miR-126a, miR-132, miR-146 and miR-155 and decreased expression of miR-20a and miR-92a were observed in the vehicle-treated ApoE-/- mice. While NR1 treatment led to a significant reduction in the expression of miR-21, miR-26a, miR-126 and increased expression of miR-20a.ConclusionCollectively, our results demonstrated for the first time the anti-atherosclerotic effects of NR1, which could be in part mediated through its multiple targeting effects on inflammation, oxidative stress, lipid metabolism and microRNA expression. These results therefore justify further evaluation of NR1 as a therapeutic agent treating atherosclerosis.
Project description:ObjectiveAtherosclerotic plaques that are prone to disruption and acute thrombotic vascular events are characterized by large necrotic cores. Necrotic cores result from the combination of macrophage apoptosis and defective phagocytic clearance (efferocytosis) of these apoptotic cells. We previously showed that macrophages with tyrosine kinase-defective Mertk receptor (Mertk(KD)) have a defect in phagocytic clearance of apoptotic macrophages in vitro. Herein we test the hypothesis that the Mertk(KD) mutation would result in increased accumulation of apoptotic cells and promote necrotic core expansion in a mouse model of advanced atherosclerosis.Methods and resultsMertk(KD);Apoe(-/-) mice and control Apoe(-/-) mice were fed a Western-type diet for 10 or 16 weeks, and aortic root lesions were analyzed for apoptosis and plaque necrosis. We found that the plaques of the Mertk(KD);Apoe(-/-) mice had a significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive apoptotic cells. Most importantly, there were more non-macrophage-associated apoptotic cells in the Mertk(KD) lesions, consistent with defective efferocytosis. The more advanced (16-week) Mertk(KD);Apoe(-/-) plaques were more necrotic, consistent with a progression from apoptotic cell accumulation to plaque necrosis in the setting of a defective efferocytosis receptor.ConclusionsIn a mouse model of advanced atherosclerosis, mutation of the phagocytic Mertk receptor promotes the accumulation of apoptotic cells and the formation of necrotic plaques. These data are consistent with the notion that a defect in an efferocytosis receptor can accelerate the progression of atherosclerosis and suggest a novel therapeutic target to prevent advanced plaque progression and its clinical consequences.
Project description:BackgroundSphingomyelin synthase 2 (SMS2) contributes to de novo sphingomyelin (SM) biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis.MethodsThe Adenovirus containing SMS2 gene was given into 10-week ApoE KO C57BL/6J mice by femoral intravenous injection. In the control group, the Adenovirus containing GFP was given. To confirm this model, we took both mRNA level examination (RT-PCR) and protein level examination (SMS activity assay).ResultWe generated recombinant adenovirus vectors containing either human SMS2 cDNA (AdV-SMS2) or GFP cDNA (AdV-GFP). On day six after intravenous infusion of 2 × 10(11) particle numbers into ten-week-old apoE KO mice, AdV-SMS2 treatment significantly increased liver SMS2 mRNA levels and SMS activity (by 2.7-fold, 2.3-fold, p < 0.001, respectively), compared to AdV-GFP treated mice. Moreover, plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and sphingomyelin (SM) levels were significantly increased by 39% (p < 0.05), 42% (p < 0.05), 68% (p < 0.001), and 45% (p < 0.05), respectively. Plasma high-density lipoprotein cholesterol (HDL-C), phosphatidylcholine (PC), and PC/SM ratio were decreased by 42% (p < 0.05), 18% (p < 0.05), and 45% (p < 0.05), respectively. On day 30, the atherosclerotic lesions on the aortic arch of AdV-SMS2 treated mice were increased, and the lesion areas on the whole aorta and in the aortic root were significantly increased (p < 0.001). Furthermore, the collagen content in the aorta root was significantly decreased (p < 0.01).ConclusionsOur results present direct morphological evidence for the pro-atherogenic capabilities of SMS2. SMS2 could be a potential target for treating atherosclerosis.
Project description:IntroductionWe previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model.MethodsAtherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE-/- background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet.ResultsWe generated an inducible SMC Notch2 null on the ApoE-/- background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet.ConclusionsOur data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.
Project description:BackgroundIt has been demonstrated that berberine (BBR), a kind of alkaloid derived from Chinese herbal medicine, has multiple pharmacological effects on human's diseases including anti-atherosclerosis action. However, although the previous studies showed that the beneficial impact of BBR on atherosclerosis might be associated with proprotein convertase subtilisin/kexin type 9 (PCSK9), the exact underlying mechanism are not fully determined. The present study aimed to investigate potential mechanisms of anti-atherosclerosis by BBR using ApoE-/- mice.MethodsThe eight-week mice were divided into five groups: group 1 (wild type C57BL/6J mice with normal diet), group 2 (ApoE-/- mice with normal diet), group 3 [ApoE-/- mice with high-fat diet (HFD)], group 4 (ApoE-/- mice with HFD, and treatment with low dose BBR of 50 mg/kg/d), and group 5 (ApoE-/- mice with HFD, and treatment with high dose BBR of 100 mg/kg/d). After a 16-week treatment, the blood sample, aorta and liver were collected for lipid analysis, hematoxylin-eosin (HE) or oil red O staining, and Western blotting respectively. Besides, HepG2 Cells were cultured and treated with different concentrations of BBR (0, 5, 25 and 50 µg/mL) for 24 hours. Subsequently, cells were collected for real-time PCR or western blotting assays. Finally, the expression levels of PCSK9, LDL receptor (LDLR), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor class B type I (SR-BI) were examined.ResultsFifty mg/kg/d and 100 mg/kg/d of BBR decreased total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDL-C) level. Moreover, BBR reduced aorta atherosclerotic plaque, and ameliorated lipid deposition in ApoE-/- mice fed with HFD. Finally, in vitro study showed that BBR promoted intracellular cholesterol efflux, up-regulated LDLR and down-regulated PCSK9 expression via the ERK1/2 pathway in cultured HepG2 cells.ConclusionsData indicated that BBR significantly attenuated lipid disorder, reduced aortic plaque formation, and alleviated hepatic lipid accumulation in ApoE-/- mice fed with HFD, which was associated with down-regulation of PCSK9 through ERK1/2 pathway.
Project description:Background and aimsCCN4/WISP-1 regulates various cell behaviours that contribute to atherosclerosis progression, including cell adhesion, migration, proliferation and survival. We therefore hypothesised that CCN4 regulates the development and progression of atherosclerotic plaques.MethodsWe used a high fat fed ApoE-/- mouse model to study atherosclerotic plaque progression in the brachiocephalic artery and aortic root. In protocol 1, male ApoE-/- mice with established plaques were given a CCN4 helper-dependent adenovirus to see the effect of treatment with CCN4, while in protocol 2 male CCN4-/-ApoE-/- were compared to CCN4+/+ApoE-/- mice to assess the effect of CCN4 deletion on plaque progression.ResultsCCN4 overexpression resulted in reduced occlusion of the brachiocephalic artery with less apoptosis, fewer macrophages, and attenuated lipid core size. The amount of plaque found on the aortic root was also reduced. CCN4 deficiency resulted in increased apoptosis and occlusion of the brachiocephalic artery as well as increased plaque in the aortic root. Additionally, in vitro cells from CCN4-/-ApoE-/- mice had higher apoptotic levels. CCN4 deficiency did not significantly affect blood cholesterol levels or circulating myeloid cell populations.ConclusionsWe conclude that in an atherosclerosis model the most important action of CCN4 is the effect on cell apoptosis. CCN4 provides pro-survival signals and leads to reduced cell death, lower macrophage number, smaller lipid core size and reduced atherosclerotic plaque burden. As such, the pro-survival effect of CCN4 is worthy of further investigation, in a bid to find a therapeutic for atherosclerosis.
Project description:Leukocytosis is associated with increased cardiovascular disease risk in humans and develops in hypercholesterolemic atherosclerotic animal models. Leukocytosis is associated with the proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) in mice with deficiencies of the cholesterol efflux-promoting ABC transporters ABCA1 and ABCG1 in BM cells. Here, we have determined the role of endogenous apolipoprotein-mediated cholesterol efflux pathways in these processes. In Apoe⁻/⁻ mice fed a chow or Western- type diet, monocytosis and neutrophilia developed in association with the proliferation and expansion of HSPCs in the BM. In contrast, Apoa1⁻/⁻ mice showed no monocytosis compared with controls. ApoE was found on the surface of HSPCs, in a proteoglycan-bound pool, where it acted in an ABCA1- and ABCG1-dependent fashion to decrease cell proliferation. Accordingly, competitive BM transplantation experiments showed that ApoE acted cell autonomously to control HSPC proliferation, monocytosis, neutrophilia, and monocyte accumulation in atherosclerotic lesions. Infusion of reconstituted HDL and LXR activator treatment each reduced HSPC proliferation and monocytosis in Apoe⁻/⁻ mice. These studies suggest a specific role for proteoglycanbound ApoE at the surface of HSPCs to promote cholesterol efflux via ABCA1/ABCG1 and decrease cell proliferation, monocytosis, and atherosclerosis. Although endogenous apoA-I was ineffective, pharmacologic approaches to increasing cholesterol efflux suppressed stem cell proliferative responses.