Diet and Atherosclerosis in Apolipoprotein E-Deficient Mice

Atherosclerosis is a multifactorial, long-lasting process in humans. Accordingly, animal models in which more rapid changes occur can be useful for the study of this process. Among such models are apolipoprotein E-deficient (apoE?/?) mice, which give insight into the human process. ApoE?/? mice show impaired clearing of plasma lipoproteins and develop atherosclerosis in a short time, and hence they are an excellent model in which to assess the impact of dietary factors. This review considers lipid metabolism and inflammation as well as nutritional constituents affecting atherosclerosis, with reference to apoE?/? mice, and discusses the mechanisms through which they act.     

Overexpression of CCN3 Inhibits Inflammation and Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice

Background

Cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed (CCN) 3 has been recently reported to play a role in regulating inflammation of vascular endothelial cells. However, the role of CCN3 in atherosclerosis, which is characterized by vascular inflammation, remains unclear.

Hypothesis and Objectives

Overexpression of CCN3 may relieve the inflammation response in and inhibit the progress of atherosclerosis. We aimed to explore the potential roles of CCN3 in inflammation in atherosclerosis.

Strategy and Main Results

In in vitro studies using cultured human aortic endothelial cells and human umbilical vein endothelial cells, CCN3 mRNA and protein expression significantly decreased in response to tumor necrosis factor-α and interleukin-1β treatments (p<0.05), when analyzed by quantitative real-time polymerase chain reaction and Western blot. Using a mouse model of atherosclerosis, the mRNA and protein levels of CCN3 decreased by 72.2% (p = 0.041) and 86.4% (p = 0.036), respectively, compared with levels in wild-type control mice, respectively. Overexpression of CCN3 by adenovirus-mediated gene overexpression decreased low-density lipoprotein cholesterol by 48.9% (p = 0.017), total cholesterol by 58.9% (p = 0.031), and triglycerides by 56.8% (p = 0.022), and it increased high-density lipoprotein cholesterol level by 2.16-fold (p = 0.039), compared with control groups. Additionally, a reduced plaque area and increased fibrous cap were observed (p<0.05). Furthermore, CCN3 overexpression decreased cell adhesion molecule-1 mRNA expression by 84.7% (p = 0.007) and intercellular adhesion molecule-1 mRNA expression by 61.2% (p = 0.044). Inflammatory factors, including matrix metalloproteinases, cyclooxygenase 2, and tissue factor also significantly (p<0.05) decreased with CCN3 overexpression in the atherosclerotic mouse model. Additionally, CCN1 and CCN2, which have been reported to be highly expressed in aortic atherosclerotic plaques, were significantly downregulated (p<0.05) by CCN3 overexpression.

Conclusion

CCN3 overexpression is associated with control of inflammatory processes and reversion of dyslipidemia in the process of atherosclerosis, which implies that CCN3 may be a promising target in the treatment of atherosclerosis.     

Specific Neurochemical Derangements of Brain Projecting Neurons in Apolipoprotein E-Deficient Mice

Abstract: Apolipoprotein E (apoE)-deficient mice provide a useful system for studying the role of apoE in neuronal maintenance and repair. Previous studies revealed specific memory impairments in these mice that are associated with presynaptic derangements in projecting forebrain cholinergic neurons. In the present study we examined whether dopaminergic, noradrenergic, and serotonergic projecting pathways of apoE-deficient mice are also affected and investigated the mechanisms that render them susceptible. The densities of nerve terminals of forebrain cholinergic projections were monitored histochemically by measurements of acetylcholinesterase activity, whereas those of the dopaminergic nigrostriatal pathway, the noradrenergic locus coeruleus cortical projection, and the raphe-cortical serotonergic tract were measured autoradiographically using radioligands that bind specifically to the respective presynaptic transporters of these neuronal tracts. The results obtained revealed that synaptic densities of cholinergic, noradrenergic, and serotonergic projections in specific brain regions of apoE-deficient mice are markedly lower than those of controls. Furthermore, the extent of presynaptic derangement within each of these tracts was found to be more pronounced the further away the nerve terminal is from its cell body. In contrast, the nerve terminal density of the dopaminergic neurons that project from the substantia nigra to the striatum was unaffected and was similar to that of the controls. The rank order of these presynaptic derangements at comparable distances from the respective cell bodies was found to be septohippocampal cholinergic > nucleus basalis cholinergic > locus coeruleus adrenergic > raphe serotonergic ? nigrostriatal dopaminergic, which interestingly is similar to that observed in Alzheimer's disease. These results suggest that two complementary factors determine the susceptibility of brain projecting neurons to apoE deficiency: pathway-specific differences and the distance of the nerve terminals from their cell body.     

肌细胞增强因子2在心力衰竭过程中的作用

心脏在长期过量负荷及神经体液系统过度激活的影响下,可发生以心肌细胞肥大、心肌纤维排列紊乱、心肌间质细胞增生及胚胎基因再表达增加为主的病理改变,从而引起心脏泵功能减退、心室扩张、心室肥厚和纤维化,最终导致心力衰竭.肌细胞增强因子2(myocyte enhancerfactor 2,MEF2)是一种特定的转录因子,其主要功能是促进肌细胞分化过程中的基因转录,在骨骼肌、心肌、平滑肌的发育过程中起介导细胞分化的作用.近年来的研究发现,在心力衰竭过程中.MEF2提供了心室重构信号转导过程中的作用靶点,可能参与了心室肥厚与心力衰竭的过程.     

Identification of Aortic Arch-Specific Quantitative Trait Loci for Atherosclerosis by an Intercross of DBA/2J and 129S6 Apolipoprotein E-Deficient Mice

The genetic background of apolipoprotein E (apoE) deficient mice influences atherosclerotic plaque development. We previously reported three quantitative trait loci (QTL), Aath1–Aath3, that affect aortic arch atherosclerosis independently of those in the aortic root in a cross between C57BL6 apoEKO mice (B6-apoE) and 129S6 apoEKO mice (129-apoE). To gain further insight into genetic factors that influence atherosclerosis at different vascular locations, we analyzed 335 F2 mice from an intercross between 129-apoE and apoEKO mice on a DBA/2J genetic background (DBA-apoE). The extent of atherosclerosis in the aortic arch was very similar in the two parental strains. Nevertheless, a genome-wide scan identified two significant QTL for plaque size in the aortic arch: Aath4 on Chromosome (Chr) 2 at 137 Mb and Aath5 on Chr 10 at 51 Mb. The DBA alleles of Aath4 and Aath5 respectively confer susceptibility and resistance to aortic arch atherosclerosis over 129 alleles. Both QTL are also independent of those affecting plaque size at the aortic root. Genome analysis suggests that athero-susceptibility of Aath4 in DBA may be contributed by multiple genes, including Mertk and Cd93, that play roles in phagocytosis of apoptotic cells and modulate inflammation. A candidate gene for Aath5 is Stab2, the DBA allele of which is associated with 10 times higher plasma hyaluronan than the 129 allele. Overall, our identification of two new QTL that affect atherosclerosis in an aortic arch-specific manner further supports the involvement of distinct pathological processes at different vascular locations.     

Butanol Extract of Acanthopanax senticosus (Rupr. et Maxim.) Harms Alleviates Atherosclerosis in Apolipoprotein E-Deficient Mice Fed a High-Fat Diet

This study investigated the effect of butanol extract of AS (ASBUE) on atherosclerosis in apolipoprotein E-deficient (ApoE^−/−) mice. The mice were administered ASBUE (390 or 130 mg/kg/day) or rosuvastatin (RSV) via oral gavage for eight weeks. In ApoE^−/− mice, ASBUE suppressed the abnormal body weight gain and improved serum and liver biochemical indicators. ASBUE remarkably reduced the aortic plaque area, improved liver pathological conditions, and lipid metabolism abnormalities, and altered the intestinal microbiota structure in ApoE^−/− mice. In the vascular tissue of ASBUE-treated mice, P-IKKβ, P-NFκB, and P-IκBα levels tended to decrease, while IκB-α increased in high fat-diet-fed atherosclerotic mice. These findings demonstrated the anti-atherosclerotic potential of ASBUE, which is mediated by the interaction between the gut microbiota and lipid metabolism and regulated via the Nuclear Factor-kappa B (NF-κB) pathway. This work paves the groundwork for subsequent studies to develop innovative drugs to treat atherosclerosis.     

Anti-Inflammatory and Anti-Thrombotic Effects of the Fungal Metabolite Galiellalactone in Apolipoprotein E-Deficient Mice

Patients suffering from chronic inflammatory diseases have an increased mortality risk resulting from cardiovascular disorders due to enhanced atherosclerotic and thrombotic events. Until now, it is not completely understood in which way an abnormal expression of pro-inflammatory mediators contributes to this elevated cardiovascular risk, but there is a need for new drugs that on the one hand suppress the expression of pro-inflammatory mediators and on the other hand inhibit arterial platelet adhesion. Thus, we analyzed the anti-inflammatory and anti-thrombotic capacity of the fungal metabolite Galiellalactone in atherosclerosis-prone apolipoprotein E-deficient mice. Treatment of the mice with Galiellalactone lowered the inflammatory expression profile and improved blood clotting times, as well as platelet adhesion to the injured common carotid artery. The results indicate that administration of Galiellalactone is able to reduce the extent of inflammation and arterial platelet adhesion in this mouse model.     

Variation in Type 2 Diabetes-Related Phenotypes among Apolipoprotein E-Deficient Mouse Strains

We recently have found that apolipoprotein E-deficient (Apoe^-/-) mice with the C57BL/6 background develop type 2 diabetes when fed a Western diet for 12 weeks. In the present study we constructed multiple Apoe^-/- mouse strains to find diabetes-related phenotyptic variations that might be linked to atherosclerosis development. Evaluation of both early and advanced lesion formation in aortic root revealed that C57BL/6, SWR/J, and SM/J Apoe^-/- mice were susceptible to atherosclerosis and that C3H/HeJ and BALB/cJ Apoe^-/- mice were relatively resistant. On a chow diet, fasting plasma glucose varied among strains with C3H/HeJ having the highest (171.1 ± 9.7 mg/dl) and BALB/cJ the lowest level (104.0 ± 6.6 mg/dl). On a Western diet, fasting plasma glucose rose significantly in all strains, with C57BL/6, C3H/HeJ and SWR/J exceeding 250 mg/dl. BALB/cJ and C3H/HeJ were more tolerant to glucose loading than the other 3 strains. C57BL/6 was sensitive to insulin while other strains were not. Non-fasting blood glucose was significantly lower in C3H/HeJ and BALB/cJ than C57BL/6, SM/J, and SWR/J. Glucose loading induced the 1st and the 2nd phase of insulin secretion in BALB/cJ, but the 2nd phase was not observed in other strains. Morphological analysis showed that BALB/cJ had the largest islet area (1,421,493 ± 61,244 μm²) and C57BL/6 had the smallest one (747,635 ± 41,798 μm²). This study has demonstrated strain-specific variations in the metabolic and atherosclerotic phenotypes, thus laying the basis for future genetic characterization.     

Pitavastatin Reduces Inflammation in Atherosclerotic Plaques in Apolipoprotein E-Deficient Mice with Late Stage Renal Disease

Objectives

Chronic renal disease (CRD) accelerates atherosclerosis and cardiovascular calcification. Statins reduce low-density lipoprotein-cholesterol levels in patients with CRD, however, the benefits of statins on cardiovascular disease in CRD remain unclear. This study has determined the effects of pitavastatin, the newest statin, on arterial inflammation and calcification in atherogenic mice with CRD.

Methods and Results

CRD was induced by 5/6 nephrectomy in cholesterol-fed apolipoprotein E-deficient mice. Mice were randomized into three groups: control mice, CRD mice, and CRD mice treated with pitavastatin. Ultrasonography showed that pitavastatin treatment significantly attenuated luminal stenosis in brachiocephalic arteries of CRD mice. Near-infrared molecular imaging and correlative Mac3 immunostaining demonstrated a significant reduction in macrophage accumulation in pitavastatin-treated CRD mice. Pitavastatin treatment reduced levels of osteopontin in plasma and atherosclerotic lesions in CRD mice, but did not produce a significant reduction in calcification in atherosclerotic plaques as assesses by histology. CRD mice had significantly higher levels of phosphate in plasma than did control mice, which did not change by pitavastatin. In vitro, pitavastatin suppressed the expression of osteopontin in peritoneal macrophages stimulated with phosphate or calcium/phosphate in concentrations similar to those found in human patients with CRD.

Conclusion

Our study provides in vivo evidence that pitavastatin reduces inflammation within atherosclerotic lesions in CRD mice.     

IL-25 Inhibits Atherosclerosis Development in Apolipoprotein E Deficient Mice

Objective

IL-25 has been implicated in the initiation of type 2 immunity and in the protection against autoimmune inflammatory diseases. Recent studies have identified the novel innate lymphoid type 2 cells (ILC2s) as an IL-25 target cell population. The purpose of this study was to evaluate if IL-25 has any influence on atherosclerosis development in mice.

Methods and Results

Administration of 1 μg IL-25 per day for one week to atherosclerosis-prone apolipoprotein (apo)E deficient mice, had limited effect on the frequency of T cell populations, but resulted in a large expansion of ILC2s in the spleen. The expansion was accompanied by increased levels of anti-phosphorylcholine (PC) natural IgM antibodies in plasma and elevated levels of IL-5 in plasma and spleen. Transfer of ILC2s to apoE deficient mice elevated the natural antibody-producing B1a cell population in the spleen. Treatment of apoE/Rag-1 deficient mice with IL-25 was also associated with extensive expansion of splenic ILC2s and increased plasma IL-5, suggesting ILC2s to be the source of IL-5. Administration of IL-25 in IL-5 deficient mice resulted in an expanded ILC2 population, but did not stimulate generation of anti-PC IgM, indicating that IL-5 is not required for ILC2 expansion but for the downstream production of natural antibodies. Additionally, administration of 1 μg IL-25 per day for 4 weeks in apoE deficient mice reduced atherosclerosis in the aorta both during initiation and progression of the disease.

Conclusions

The present findings demonstrate that IL-25 has a protective role in atherosclerosis mediated by innate responses, including ILC2 expansion, increased IL-5 secretion, B1a expansion and natural anti-PC IgM generation, rather than adaptive Th2 responses.     

Vitamin D Deficiency Induces High Blood Pressure and Accelerates Atherosclerosis in Mice

Multiple epidemiological studies link vitamin D deficiency to increased cardiovascular disease (CVD), but causality and possible mechanisms underlying these associations are not established. To clarify the role of vitamin D-deficiency in CVD in vivo, we generated mouse models of diet-induced vitamin D deficiency in two backgrounds (LDL receptor- and ApoE-null mice) that resemble humans with diet-induced hypertension and atherosclerosis. Mice were fed vitamin D-deficient or -sufficient chow for 6 weeks and then switched to high fat (HF) vitamin D-deficient or –sufficient diet for 8–10 weeks. Mice with diet-induced vitamin D deficiency showed increased systolic and diastolic blood pressure, high plasma renin, and decreased urinary sodium excretion. Hypertension was reversed and renin was suppressed by returning chow-fed vitamin D-deficient mice to vitamin D-sufficient chow diet for 6 weeks. On a HF diet, vitamin D-deficient mice had ∼2-fold greater atherosclerosis in the aortic arch and ∼2–8-fold greater atherosclerosis in the thoracic and abdominal aorta compared to vitamin D-sufficient mice. In the aortic root, HF-fed vitamin D-deficient mice had increased macrophage infiltration with increased fat accumulation and endoplasmic reticulum (ER) stress activation, but a lower prevalence of the M1 macrophage phenotype within atherosclerotic plaques. Similarly, peritoneal macrophages from vitamin D-deficient mice displayed an M2-predominant phenotype with increased foam cell formation and ER stress. Treatment of vitamin D-deficient mice with the ER stress reliever PBA during HF feeding suppressed atherosclerosis, decreased peritoneal macrophage foam cell formation, and downregulated ER stress proteins without changing blood pressure. Thus, we suggest that vitamin D deficiency activates both the renin angiotensin system and macrophage ER stress to contribute to the development of hypertension and accelerated atherosclerosis, highlighting vitamin D replacement as a potential therapy to reduce blood pressure and atherosclerosis.     

Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β₃-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.     

MEF2A基因突变对血管平滑肌细胞增殖迁移及其表型的影响

目的:观察肌细胞增强因子2A(MEF2A)基因突变对血管平滑肌细胞(VSMC)增殖迁移及其表型的影响。方法:分别将野生型(WT)MEF2A质粒(WT组)、21个核苷酸缺失突变型(△21,显性负突变)MEF2A质粒(△21组)以及MEF2A siRNA(siRNA组)转染进人主动脉血管平滑肌细胞(VSMC),通过溴化噻唑基蓝四唑(MTT)法和Millicell小室观察各组VSMC的增殖和迁移变化,免疫印迹(Western blotting)检测各组VSMC之间MEF2A蛋白、平滑肌α肌动蛋白(α-SM-actin)、SM22α、骨桥蛋白和丝裂素活化蛋白激酶(MAPK)信号通路表达差异。结果:MEF2A△21组和MEF2A siRNA组的VSMC增殖增加,迁移数量增多;同时此两组中α-SM-actin和SM22α表达减少,骨桥蛋白表达增加;磷酸化p38和ERK1/2表达也明显增强。结论:MEF2A基因显性负突变及沉默可使VSMC向合成型转化,其增殖和迁移能力增加。而p38和ERK1/2MAPK信号通路可能参与MEF2A基因介导的血管平滑肌细胞表型转化。     

Site-Specific Dephosphorylation of Tau of Apolipoprotein E-Deficient and Control Mice by M1 Muscarinic Agonist Treatment

Abstract : Apolipoprotein E (apoE)-deficient mice have memory deficits that are associated with synaptic loss of basal forebrain cholinergic projections and with hyperphosphorylation of distinct epitopes of the microtubuleassociated protein tau. Furthermore, treatment of apoEdeficient mice with the M1 selective agonist 1-methylpiperidine-4-spiro-(2'-methylthiazoline) [AF 150(S)] abolishes their memory deficits and results in recovery of their brain cholinergic markers. In the present study, we used a panel of anti-tau monoclonal antibodies to further map the tau epitopes that are hyperphosphorylated in apoE-deficient mice and examined the effects of prolonged treatment with AF 150(S). This revealed that tau of apoE-deficient mice contains a distinct, hyperphosphorylated "hot spot" domain which is localized N-terminally to the microtubule binding domain of tau, and that AF150(S) has an epitope-specific tau dephosphorylating effect whose magnitude is affected by apoE deficiency. Accordingly, epitopes which reside in the hyperphosphorylated "hot spot" are dephosphorylated by AF 150(S) in apoE-deficient mice but are almost unaffected in the controls, whereas epitopes which flank this tau domain are dephosphorylated by AF150(S) in both mice groups. In contrast, epitopes located at the N and C terminals of tau are unaffected by AF150(S) in both groups of mice. These findings suggest that apoE deficiency results in hyperphosphorylation of a distinct tau domain whose excess phosphorylation can be reduced by muscarinic treatment.