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.     

Adiponectin Receptor 2 Deficiency Results in Reduced Atherosclerosis in the Brachiocephalic Artery in Apolipoprotein E Deficient Mice

Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2. The original aim of this study was to investigate the effect of combined AdipoR1 and AdipoR2 deficiency (AdipoR1^-/-AdipoR2^-/-) on atherosclerosis. However, we made the interesting observation that AdipoR1 ^-/- AdipoR2 ^-/- leads to embryonic lethality demonstrating the critical importance of the adiponectin signalling system during development. We then investigated the effect of AdipoR2-ablation on the progression of atherosclerosis in apolipoprotein E deficient (ApoE ^-/-) mice. AdipoR2^-/-ApoE^-/- mice fed an atherogenic diet had decreased plaque area in the brachiocephalic artery compared with AdipoR2 ^+/+ApoE^-/- littermate controls as visualized in vivo using an ultrasound biomicroscope and confirmed by histological analyses. The decreased plaque area in the brachiocephalic artery could not be explained by plasma cholesterol levels or inflammatory status. However, accumulation of neutral lipids was decreased in peritoneal macrophages from AdipoR2^-/-ApoE^-/- mice after incubation with oxidized LDL. This effect was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages from AdipoR2^-/-ApoE^-/- mice compared with AdipoR2^+/+ApoE^-/- controls after incubation with oxidized LDL. In summary, we show that adiponectin receptors are crucial during embryonic development and that AdipoR2-deficiency slows down the progression of atherosclerosis in the brachiocephalic artery of ApoE-deficient mice.     

Association Between Apolipoprotein E Gene Polymorphism and Myocardial Infarction

We investigated apolipoprotein E (APOE) genotypic influence on myocardial infarction risk in South India, where the disease is emerging as a major threat to the public health care system. The study included 412 subjects: 202 myocardial infarction patients and 210 age- and sex-matched controls. DNA was isolated, the polymorphism of the APOE gene was subjected to PCR, and lipid levels were evaluated. The prevalence of E3/E4 genotypes in patients (18.3%) was 1.5-fold that of controls (11.0%, p < 0.05), and the prevalence of E2/E3 genotypes was higher in controls (6.7%) than in patients (4%). The ε4 allele was significantly associated with myocardial infarction: χ ² = 12.4; OR 2.2 (CI 95%: 1.4–3.4), p < 0.004, for ε4 versus ε3 and χ ² = 5.7; OR 2.7 (CI 95%: 1.1–6.5), p > 0.01, for ε4 versus ε2. A significant association of the ε4 allele, especially the E3/E4 genotype, with myocardial infarction was observed.     

Rapid and Efficient Production of Coronary Artery Ligation and Myocardial Infarction in Mice Using Surgical Clips

Aims

The coronary artery ligation model in rodents mimics human myocardial infarction (MI). Normally mechanical ventilation and prolonged anesthesia period are needed. Recently, a method has been developed to create MI by popping-out the heart (without ventilation) followed by immediate suture ligation. Mortality is high due to the time-consuming suture ligation process while the heart is exposed. We sought to improve this method and reduce mortality by rapid coronary ligation using a surgical clip instead of a suture.

Methods and Results

Mice were randomized into 3 groups: clip MI (CMI), suture MI (SMI), or sham (SHAM). In all groups, heart was manually exposed without intubation through a small incision on the chest wall. Unlike the conventional SMI method, mice in the CMI group received a metal clip on left anterior descending artery (LAD), quickly dispensed by an AutoSuture Surgiclip^™. The CMI method took only 1/3 of ligation time of the standard SMI method and improved post-MI survival rate. TTC staining and Masson’s trichrome staining revealed a similar degree of infarct size in the SMI and CMI groups. Echocardiograph confirmed that both SMI and CMI groups had a similar reduction of ejection fraction and fraction shortening over the time. Histological analysis showed that the numbers of CD68⁺ macrophages and apoptotic cells (TUNEL-positive) are indistinguishable between the two groups.

Conclusion

This new method, taking only less than 3 minutes to complete, represents an efficient myocardial infarction model in rodents.     

Polymorphism of the Apolipoprotein E Gene and Risk of Myocardial Infarction

The apolipoprotein E (ApoE) gene polymorphism resulting from nucleotide substitutions in exon 4 was analyzed in Russian and Tatar patients with myocardial infarction (MI) from Bashkortostan. Alleles 2, 3, and 4 were identified by PCR. The genotype frequency distribution proved to be age-dependent in healthy Russians, genotype 2/3 increasing in frequency in subjects over 45. Russians who suffered MI under 45 had lower frequencies of genotype E3/3 (50.00% vs. 75.47% in controls of the same age, = 0.013, OR = 0.33) and allele 3 (72.12% vs. 85.85%, = 0.020, OR = 0.43) and a higher frequency of allele 4 (22.12% vs. 10.38%, = 0.030, OR = 2.45). Russians who suffered MI complicated by cardiogenic shock (CS) had a significantly higher frequency of genotype 3/4 and lower frequencies of genotype 3/3 and allele 3 as compared with MI patients without CS. In Tatars, genotype 4/4 occurred at a frequency of 14.29% in patients who suffered MI under 45, and was not detected in healthy subjects of the same age ( = 0.024, OR = 17.85). Thus, the ApoE polymorphism was associated with higher risk of MI in Russians and Tatars under 45.     

Meta-Analysis of Apolipoprotein E Gene Polymorphism and Susceptibility of Myocardial Infarction

A number of case-control studies have been conducted to clarify the association between ApoE polymorphisms and myocardial infarction (MI); however, the results are inconsistent. This meta-analysis was performed to clarify this issue using all the available evidence. Searching in PubMed retrieved all eligible articles. A total of 33 studies were included in this meta-analysis, including 18752 MI cases and 18963 controls. The pooled analysis based on all included studies showed that the MI patients had a decreased frequency of the ε2 allele (OR = 0.78, 95% CI = 0.70–0.87) and an increased frequency of the ε4 allele (OR = 1.15, 95% CI = 1.10–1.20); The results also showed a decreased susceptibility of MI in the ε2ε3 vs. ε3ε3 analysis (OR = 0.79, 95% CI = 0.68–0.90) and in the ε2 vs. ε3 analysis (OR = 0.78, 95% CI = 0.69–0.89), an increased susceptibility of MI in the ε3ε4 vs. ε3ε3 analysis (OR = 1.26, 95% CI = 1.12–1.41), in the ε4 vs. ε3 analysis (OR = 1.22, 95% CI = 1.12–1.32) and in the ε4ε4 vs. ε3ε3 analysis (OR = 1.59, 95% CI = 1.15–2.19). However, there were no significant associations among polymorphisms and MI for the following genetic models: frequency of the ε3 allele (OR = 0.99, 95% CI = 0.96–1.02); ε2ε2 vs. ε3ε3 analysis (OR = 0.73, 95% CI = 0.40–1.32); or ε2ε4 vs. ε3ε3 analysis (OR = 1.10, 95% CI = 0.99–1.21). Our results suggested that the ε4 allele of ApoE is a risk factor for the development of MI and the ε2 allele of ApoE is a protective factor in the development of MI.     

SR-BI in Bone Marrow Derived Cells Protects Mice from Diet Induced Coronary Artery Atherosclerosis and Myocardial Infarction

SR-BI deficient mice that are also hypomorphic for apolipoprotein E expression develop diet induced occlusive coronary artery atherosclerosis, myocardial infarction and early death. To test the role of SR-BI in bone marrow derived cells, we used bone marrow transplantation to generate SR-BI-null; apoE-hypomorphic mice in which SR-BI expression was restored solely in bone marrow derived cells. SR-BI-null; apoE-hypomorphic mice were transplanted with SR-BI^+/+apoE-hypomorphic, or control, autologous SR-BI-null; apoE-hypomorphic bone marrow. Four weeks later, mice were fed a high-fat, high-cholesterol, cholate-containing diet to induce coronary artery atherosclerosis. Mice transplanted with autologous bone marrow developed extensive aortic atherosclerosis and severe occlusive coronary artery atherosclerosis after 4 weeks of feeding. This was accompanied by myocardial fibrosis and increased heart weights. In contrast, restoration of SR-BI expression in bone marrow derived-cells reduced diet induced aortic and coronary artery atherosclerosis, myocardial fibrosis and the increase in heart weights in SR-BI-null; apoE-hypomorphic mice. Restoration of SR-BI in bone marrow derived cells did not, however, affect steady state lipoprotein cholesterol levels, but did reduce plasma levels of IL-6. Monocytes from SR-BI-null mice exhibited a greater capacity to bind to VCAM-1 and ICAM-1 than those from SR-BI^+/+ mice. Furthermore, restoration of SR-BI expression in bone marrow derived cells attenuated monocyte recruitment into atherosclerotic plaques in mice fed high fat, high cholesterol cholate containing diet. These data demonstrate directly that SR-BI in bone marrow-derived cells protects against both aortic and CA atherosclerosis.     

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available ^1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important ^{7, 14, 30, 39}. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP ³⁹. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds³⁹. In addition to infarct staining, we tested cardiac troponin I (cTnI) as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction^{6, 8, 30, 40, 41}. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.     

The Effect of Soluble RAGE on Inhibition of Angiotensin II-Mediated Atherosclerosis in Apolipoprotein E Deficient Mice

Background

The cross talk between RAGE and angiotensin II (AngII) activation may be important in the development of atherosclerosis. Soluble RAGE (sRAGE), a truncated soluble form of the receptor, acts as a decoy and prevents the inflammatory response mediated by RAGE activation. In this study, we sought to determine the effect of sRAGE in inhibiting AngII-induced atherosclerosis in apolipoprotein E knockout mice (Apo E KO).

Methods and Results

9 week old Apo E KO mice were infused subcutaneously with AngII (1 µg/min/kg) and saline for 4 weeks using osmotic mini-pumps. The mice were divided into 4 groups 1. saline infusion and saline injection; 2. saline infusion and sRAGE injection; 3. AngII infusion and saline injection; 4. AngII infusion and sRAGE injection. Saline or 0.5 µg, 1 µg, to 2 µg/day/mouse of sRAGE were injected intraperitoneally daily for 28 days. We showed that atherosclerotic plaque areas in the AngII-infused Apo E KO mice and markers of inflammation such as RAGE, ICAM-1, VCAM-1, and MCP-1 were increased in aorta compared to that of the Apo E KO mice. However, the treatment of 0.5 µg, 1 µg, and 2 µg of sRAGE in AngII group resulted in the dose-dependent decrease in atherosclerotic plaque area. We also demonstrated that sRAGE decreased RAGE expression level as well as inflammatory cytokines and cell adhesion molecules in AngII or HMGB1 treated-rat aorta vascular smooth muscle cells.

Conclusion

The results demonstrated that partical blockade of RAGE activation by sRAGE prevent AngII -induced atherosclerosis. Therefore these results suggested that first, RAGE activation may be important in mediating AngII-induced atherogenesis, and second, AngII activation is a major pathway in the development of atherosclerosis. Taken together, results from this study may provide the basis for future anti- atherosclerotic drug development mediated through RAGE activation.     

冠脉结扎和介入两种方法制备犬心肌梗死模型的比较

目的观察开胸结扎冠状动脉与闭胸明胶海绵栓塞法制备急性心肌梗死（AMI）动物模型的特点。方法分别经开胸结扎犬冠状动脉左前降支主干及闭胸冠脉栓塞的方法阻断冠脉血流;采用单级肢体导联和胸导联方式,在阻断前后监测心电图波形变化;造模72h后取心肌组织行病理切片染色。结果经心电图和病理验证,两种方法均可成功制备犬心梗模型,开胸冠脉结扎犬死亡率较高,而冠脉栓塞成活率高。结论相较开胸冠脉结扎法,闭胸栓塞法制备心梗模型对动物损伤小,成活率高,具推广价值。     

心导管介入冠状动脉封堵兔急性心肌梗死模型的建立

目的应用心导管介入方法封堵冠状动脉制备兔急性心肌梗死模型。方法选择雄性新西兰兔,先行冠状动脉造影,利用导引钢丝将微导管置于左前降支远端,将高分子栓塞剂与碘油混合配制成封闭胶,经微导管注入血管,造成急性心肌梗死。术前、术中和术后l周记录心电图变化。实验终点切取心肌组织标本分别行苏木素一伊红（H．E）染色、氯化硝基四氮唑蓝（NBT）染色、免疫组化染色。结果造模动物20只,存活16只。冠脉造影显示封闭胶持续滞留于左前降支远端,提示血管完全堵塞。心电图提示存在动态变化,ST段抬高,病理性Q波逐渐形成。心脏大体观测提示左心室前侧壁呈灰白色为梗死区。E染色提示梗死区局部纤维组织增生、疤痕形成、钙盐沉积,缺血区肌束变性、炎症细胞浸润,符合典型心肌梗死的病理变化。NBT染色后测定梗死面积为28．32％±5．21％。免疫组化染色提示缺血区CD34阳性面积和血管新生密度明显高于梗死区及正常组织区（P〈0．05）。结论通过心导管介入方法制备兔急性心肌梗死模型成功,避免了开胸损伤对实验结果的影响,更符合临床急性心肌梗死的病理特点。     

RDW和hs-CRP水平在急性心肌梗死中的表达及与冠状动脉狭窄程度的关系

目的:研究红细胞分布宽度(RDW)和高敏C反应蛋白(hs-CRP)水平在急性心肌梗死中的表达及与冠状动脉狭窄程度的关系。方法:选取2010年1月到2015年1月我院收治的急性心肌梗死患者300例(研究组),另选取单纯心绞痛患者300例(对照组),比较两组RDW、hs-CRP、Gensini评分和冠状动脉病变支数,并分析RDW、hs-CRP和Gensini评分、冠状动脉病变支数的关系。结果:研究组RDW、hs-CRP、Gensini评分和冠状动脉病变支数均显著高于对照组,两组比较差异具有统计学意义(P0.05);冠状动脉Gensini评分和病变支数与RDW、hs-CRP呈正相关关系(r=0.58,0.69,0.49,0.57,P0.05),同时RDW和hs-CRP呈正相关关系(P0.05)。结论:急性心肌梗死患者会出现RDW和hs-CRP水平增高现象,和冠状动脉狭窄程度呈正相关关系。     

Association of Polymorphic Marker A1/A2 of Gene ITGB3 with Coronary Artery Disease and Myocardial Infarction

The distributions of the alleles and genotypes of polymorphic marker A1/A2 of gene ITGB3 encoding the 3 subunit of integrin IIb3 in groups of patients with coronary artery disease (CAD), including those who have had myocardial infarction (MI), and in the control group have been compared. Analysis using Fisher's test has not revealed significant differences between these groups with respect to the distributions of the alleles and genotypes of polymorphic marker A1/A2 of gene ITGB3. This indicates that this polymorphic marker is not associated with either CAD or MI in the Moscow population.     

Apolipoprotein E4 Is Deficient in Inducing Macrophage ABCA1 Expression and Stimulating the Sp1 Signaling Pathway

ATP binding cassette A1 (ABCA1) is a membrane protein that promotes cellular cholesterol efflux. Using RAW 264.7 macrophages, we studied the relative effects of apolipoprotein (apo) E3 and apoE4 on ABCA1 and on the signaling pathway that regulates its expression. Both lipid-associated and lipid-free apoE4 forms induced ∼30% lower levels of ABCA1 protein and mRNA than apoE3 forms. Phosphorylated levels of phosphoinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ) and specificity protein 1 (Sp1) were also lower when treated with apoE4 compared to apoE3. The reduced ability of apoE4 to induce ABCA1 expression, PKCζ and Sp1 phosphorylation were confirmed in human THP-1 monocytes/macrophages. Sequential phosphorylation of PI3K, PKCζ and Sp1 has been suggested as a mechanism for upregulation of ABCA1 expression. Both apoE3 and apoE4 reduced total cholesterol and cholesterol esters in lipid-laden RAW 264.7 cells, and induced apoAI-mediated cholesterol efflux. However, the cholesterol esters and cholesterol efflux in apoE4-treated cells were ∼50% and ∼24% lower, respectively, compared to apoE3-treated cells. Accumulation of cholesterol esters in macrophages is a mechanism for foam cell formation. Thus the reduced ability of apoE4 to activate the PI3K-PKCζ-Sp1 signaling pathway and induce ABCA1 expression likely impairs cholesterol ester removal, and increases foam cell formation.     

ApoE基因缺陷小鼠的心肌肥厚及辛伐他汀的干预研究

目的:观察高胆固醇喂养的不同周龄ApoE基因缺陷(ApoE-/-)小鼠心肌细胞和心肌间质成分的改变,并观察辛伐他汀对其的影响.方法:36只8周龄雄性ApoE-/-小鼠饲以高胆固醉饲料喂养8周即至16周龄,随机被分为三组继续喂养至24周龄组、32周龄组和40周龄组,每一周龄组为12只,再随机分为模型组6只和辛伐他汀干预组6只(25mg/kg/d),相同周龄的C57BL/6J小鼠设为对照.分别在24周、32周、40周结束时处死小鼠.常规检测血浆胆固醇水平,留取新鲜心脏组织测定总胆固醉及一氧化氮(NO)、超氧化物歧化酶(SOD)、丙二醛(MDA):另取心脏组织固定,石蜡切片,HE染色观察各组小鼠心肌细胞的变化,Masson染色观察心肌胶原改变.结果:24、32和40周龄模型组ApoE-/-小鼠血浆、心脏组织胆固醇和MDA水平逐渐增加(p<0.05),NO和SOD水平逐渐降低(p<0.05),心肌细胞直径和心肌胶原含量逐渐增加(p<0.05).与相同周龄模型组相比,辛伐他汀干预组血浆、心脏组织胆固醇和MDA水平明显降低(p<0.05),心肌细胞直径明显减小;40周龄辛伐他汀干预组左室壁平均厚度明显降低(p<0.05),32周龄和40周龄辛伐他汀干预组心肌胶原含量明显减少(p<0.05).结论:高胆固醇喂养的ApoE基因缺陷小鼠,随着周龄增加、胆固醇水平增加,抗氧化能力降低,心肌细胞直径和心肌胶原含量显著增加,辛伐他汀可能减轻心脏重构.     

经股动脉明胶海绵栓塞介入法建立犬精确局灶急性心梗模型

目的:探讨自制明胶海绵栓子经股动脉栓塞介入术制作稳定的犬精确局灶急性心肌梗死模型的可行性.方法:在数字减影血管造影(DSA)引导下,对12只杂种犬用明胶海绵颗粒经微导管栓塞左前降支(LAD)第二对角支远端分支,致前室壁或下壁缺血/梗死;于术前、术后行冠脉造影、心电图、99mTcN-MPO单光子发射型计算机断层(SPECT)心肌灌注显像,术前2h及术后1、3、6、12、24h检测肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、谷草转氨酶(AST)、肌钙蛋白I(cTnI);4W后动物处死,苏木素-伊红(HE)染色病理学确认梗死.结果:介入栓塞LAD远端分支后,2只犬由于出现室性纤颤而死亡,10只犬心梗后24h存活,连续观察28d后依然存活良好.与自身对照相比,心梗后冠脉造影示LAD远端分支闭塞;心电图VI-V3,Ⅱ,Ⅲ,aVF表现为S-T段抬高;99mTcN-MPO SPECT心肌断层显像示心梗区放射性核素摄取明显减低或缺损;AST、CK、CK.MB、LDH、cTnl均于术后升高;HE染色示心肌细胞核消失、碎裂,胞质均质红染,间质水肿.结论:微创介入法制作犬精确局灶心梗模型,具有良好的可重复性与安全性,可以为临床研究急性心肌梗死提供可靠的技术平台.     

Loss of Ahi1 Impairs Neurotransmitter Release and Causes Depressive Behaviors in Mice

Major depression is becoming one of the most prevalent forms of psychiatric disorders. However, the mechanisms of major depression are still not well-understood. Most antidepressants are only effective in some patients and produce some serious side effects. Animal models of depression are therefore essential to unravel the mechanisms of depression and to develop novel therapeutic strategies. Our previous studies showed that Abelson helper integration site-1 (Ahi1) deficiency causes depression-like behaviors in mice. In this study, we characterized the biochemical and behavioral changes in Ahi1 knockout (KO) mice. In Ahi1 KO mice, neurotransmitters including serotonin and dopamine were significantly decreased in different brain regions. However, glutamate and GABA levels were not affected by Ahi1 deficiency. The antidepressant imipramine attenuated depressive behaviors and partially restored brain serotonin level in Ahi1 KO mice. Our findings suggest that Ahi1 KO mice can be used for studying the mechanisms of depression and screening therapeutic targets.