Tanshinone II-A attenuates and stabilizes atherosclerotic plaques in apolipoprotein-E knockout mice fed a high cholesterol diet

Tanshinone II-A (Tan), a bioactive diterpene isolated from Salvia miltiorrhiza Bunge (Danshen), possesses anti-oxidant and anti-inflammatory activities. The present study investigated whether Tan can decrease and stabilize atherosclerotic plaques in Apolipoprotein-E knockout (ApoE(-/-)) mice maintained on a high cholesterol diet (HCD). Six week-old mice challenged with a HCD were randomly assigned to 4 groups: (a) C57BL/6J; (b) ApoE(-/-); (c) ApoE(-/-)+Tan-30 (30 mg/kg/d); (d) ApoE(-/-)+Tan-10 (10mg/kg/d). After 16 weeks of intervention, Tan treated mice showed decreased atherosclerotic lesion size in the aortic sinus and en face aorta. Furthermore, immunohistochemical analysis revealed that Tan rendered the lesion composition a more stable phenotype as evidenced by reduced necrotic cores, decreased macrophage infiltration, and increased smooth muscle cell and collagen contents. Tan also significantly reduced in situ superoxide anion production, aortic expression of NF-κB and matrix metalloproteinase-9 (MMP-9). In vitro treatment of RAW264.7 macrophages with Tan significantly suppressed oxidized LDL-induced reactive oxygen species production, pro-inflammatory cytokine (IL-6, TNF-α, MCP-1) expression, and MMP-9 activity. Tan attenuates the development of atherosclerotic lesions and promotes plaque stability in ApoE(-/-) mice by reducing vascular oxidative stress and inflammatory response. Our findings highlight Tan as a potential therapeutic agent to prevent atherosclerotic cardiovascular diseases.     

黄连提取物对动脉硬化小鼠斑块胶原类型及MMP-9/TIMP-1 比值 的影响*

目的:探讨黄连提取物对高脂喂养ApoE-/-小鼠主动脉AS斑块内胶原类型及基质金属蛋白酶-9( MMP-9)与基质金属蛋白酶组织抑制剂( TIMP-1)比值的影响,探讨黄连提取物稳定斑块的可能作用机制.方法:33只6-8周龄的ApoE基因敲除小鼠予高脂喂养13周后,待其形成成熟的AS斑块后,随机分为3组:模型组、黄连提取物组、辛伐他汀组(阳性对照组),每组11只.继续高脂喂养,并按体重比折算给予小鼠临床推荐剂量的相应药物治疗13周,处死动物,每只小鼠取主动脉根部的4个切面,行天狼猩红染色,检测各组小鼠主动脉斑块内Ⅰ、Ⅲ型胶原含量,以及斑块内MMP-9和TIMP-1的表达,计算MMP-9/TIMP-1比值.结果:给药13周后,图像分析结果显示,黄连提取物组小鼠主动脉斑块内Ⅰ型胶原含量与模型组比较有所增加,但无显著差异(P＞0.05);辛伐他汀组和黄连提取物组小鼠主动脉斑块内Ⅲ型胶原含量与模型组比较显著降低(P＜0.01).Ⅲ型/Ⅰ型胶原比值,两给药组与模型组比较均显著降低(P＜0.01).与模型组比较,黄连提取物和辛伐他汀组小鼠主动脉斑块内MMP-9的阳性表达均明显减少(P＜0.01),黄连提取物组主动脉斑块内TIMP-1的阳性表达与模型组相比明显增加(P＜0.01),辛伐他汀组TIMP-1表达有所增加,但无统计学差异(P＞0.05),两给药组之间比较无显著差异(P＞0.05).各给药组中MMP-9/TIMP-1比值均有所降低,与模型组比较具有显著差异(P＜0.05,P＜0.01).结论:在临床推荐剂量下,黄连提取物可明显改善ApoE-/-小鼠主动脉AS斑块内胶原类型,调整斑块内MMP-9/TIMP-1比值,从而促进斑块稳定.     

液氮冻伤术建立动脉粥样硬化破裂斑块及血栓模型

目的创建一种操作简单、经济实用的动脉粥样硬化（AS）破裂斑块及血栓动物模型。方法21只雄性纯种新西兰白兔随机分为两组：液氮冻伤＋高脂喂养组（A组=11只）和高脂喂养组（B组=10只）。A组实施右颈总动脉内膜液氮冻伤术结合高脂饲料喂养,B组单纯给予高脂饲料喂养。8周末以液氮激发斑块破裂,激发前后分别采血检测血脂、hsC-RP、MMP-9及PAI-1水平;激发48h后处死所有动物,取出右颈总动脉作HE染色及免疫组化染色等,光镜及电镜观察破裂斑块及血栓形成情况。结果8周后兔血脂水平明显升高;激发后血浆hsC-RP、MMP-9及PAI-1均明显升高;所有A组兔子的右颈总动脉均可见AS破裂斑块及血栓形成,而B组兔子未见斑块或血栓形成;所建立的破裂斑块在组织结构、细胞构成、生长特征和脂质沉积方面与人类斑块相似。结论液氮冻伤术能简便、快速、高效地建立AS破裂斑块及血栓模型,从而为研究人类AS破裂斑块及血栓形成的机理和药物干预治疗提供了一种新型动物模型。     

丹参酮对于阿尔茨海默症大鼠颞叶iNOS，MMP-2 表达的 影响及机理研究

目的：应用中药丹参酮(tanshinone II A,Tan II A)治疗AD大鼠,观察TanⅡ A 干预前后,AD大鼠学习记忆、颞叶中诱导型一氧 化氮合成酶(iNOS)、基质金属蛋白酶（MMP-2）表达的变化。方法：采用beta- 淀粉样蛋白（A beta）定向注射法建立AD大鼠模型,并使用 Tan II A 干预,通过避暗测试、real-time PCR和Western Blot 分别观察大鼠学习记忆能力、大鼠颞叶MMP-2、iNOS 两者的mRNA 及蛋白表达的变化。应用SPSS13.0 进行统计学分析。结果：与假手术组相比,AD 组的平均潜伏期缩短（P＜0.01）,平均错误次数 增加（P＜0.01）,差异均有统计学意义。颞叶内iNOS、MMP-2 mRNA 表达均显著增高（P＜ 0.01, P＜0.01）;两蛋白的表达均显著增 高（P＜0.01, P＜0.01）。与AD组相比,Tan IIA 组的平均潜伏期延长（P＜0.01）,平均错误次数减少（P＜0.01）,差异均有统计学意 义。颞叶内iNOS、MMP-2 mRNA表达均显著下降（P＜0.05, P＜0.05）,两蛋白的表达均显著下降（P＜0.01, P＜0.01）。结论：Tan II A 干预可显著降低AD 大鼠颞叶中iNOS、MMP-2 mRNA及蛋白的表达,显著改善AD 大鼠的学习记忆能力。其作用机制可能是 通过降低A-beta诱导的iNOS及MMP-2 的表达,抑制氧化应激损伤来完成。     

睾酮对雄兔hs-CRP、MMP-9、HO-1、APN和动脉粥样硬化斑块稳定性的影响

目的探讨睾酮对雄兔血高敏C反应蛋白（hs-CRP）、基质金属蛋白酶-9（MMP-9）、血红素氧化酶-1（HO-1）、脂联素（APN）和动脉粥样硬化斑块稳定性的影响。方法将66只雄性新西兰纯种兔随机分为4组,其中正常对照组10只（A组）、喂以普通饲料;假去势＋球囊损伤颈动脉组16只（B组）、去势＋球囊损伤颈动脉＋生理剂量睾酮（6mg/（kg.2周））肌注组16只（C组）、去势＋球囊损伤颈动脉＋生理盐水肌注组24只（D组）均喂以高脂饲料。在实验第8周随机抽取处死部分动物,观察颈动脉病变的形态特征,抽血测睾酮、hs-CRP、MMP-9、HO-1、APN水平。结果D组兔血睾酮水平显著降低;B,C组较D组兔血hs-CRP、MMP-9水平显著降低,HO-1、APN水平显著升高;且B,C组较D组兔动脉粥样斑块的面积和内-中膜厚度（IMT）显著减小,斑块纤维帽增厚、胶原含量明显增加。结论生理剂量睾酮可以影响雄兔血hs-CRP、MMP-9、HO-1、APN水平,并能调节动脉粥样硬化斑块进展与斑块的稳定性。     

Differential induction of gelatinase B (MMP-9) and gelatinase A (MMP-2) in T lymphocytes upon alpha(4)beta(1)-mediated adhesion to VCAM-1 and the CS-1 peptide of fibronectin

Integrin alpha(4)beta(1) on the surface of T lymphocytes interacts with vascular cell adhesion molecule-1 (VCAM-1) and fibronectin during migration of lymphocytes from the blood to sites of inflammation. Migrating lymphocytes actively modify their environment through a number of mechanisms including proteolysis of the extracellular matrix by matrix metalloproteinases (MMP) synthesized by the cells. In this study, expression of MMP upon alpha(4)beta(1)-mediated adhesion of leukocytes to two major ligands, the IIICS-1 domain of fibronectin and VCAM-1, has been examined. Adhesion of T lymphoblastoid Jurkat cells to the CS-1 peptide induced expression of mRNA for two MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9). As evaluated by relative RT-PCR and Northern blot analyses, the level of mRNA was upregulated about 4- to 5-fold for both MMPs compared to control cells maintained in suspension. With time, both enzymes were detected in conditioned media and inside the cells, and their identities were verified by Western blotting and gelatin zymography. Adhesion of Jurkat cells to the second major alpha(4)beta(1) ligand, VCAM-1, upregulated mRNA for MMP-2 (3.5-fold) and failed to induce expression of mRNA for MMP-9. Accordingly, only MMP-2 protein was detected in conditioned media of cells adherent to VCAM-1. Occupancy of alpha(4)beta(1) on the surface of suspended cells with soluble CS-1 peptide or VCAM-1 did not upregulate synthesis and release of MMPs. A similar pattern of induction of MMPs after adhesion to CS-1 and VCAM-1 was observed in T lymphocytes isolated from human blood. These results demonstrate that adhesion of T lymphocytes through alpha(4)beta(1) to different ligands, which bind to similar or overlapping sites in the integrin, induces intracellular events leading to distinct patterns of MMPs biosynthesis.     

Serum gelatinases (MMP-2 and MMP-9) and VCAM-1 as a guideline in a therapeutic approach in Graves' ophthalmopathy

INTRODUCTION: The aim of this study was to estimate the influence of corticosteroids on soluble MMP-2, MMP-9 and VCAM-1 in patients with Graves ophthalmopathy (GO) in order to assess their usefulness as a guideline in a therapeutic approach. MATERIAL AND METHODS: Serum gelatinases and VCAM-1 were detected in three groups of subjects: 20 patients with GO (CAS > or = 3, anamnesis of GO > or = 1 yr), 12 patients with no clinical symptoms of ophthalmopathy (Gd) and 10 healthy volunteers. Corticosteroid therapy consisted of intravenous infusions (2 series, 3 grams each time) of methylprednisolone (MP) and subsequent treatment with oral prednisone (60 mg per day) in a tapering schedule. The serum samples were collected 24 hours before MP, 24 hours after MP, after 14 days of treatment with prednisone and at the end of corticosteroid therapy. The levels of soluble MMP-2, MMP-9 and VCAM-1 were determined by the ELISA method. RESULTS: We have found no differences in serum MMP-2 between the groups studied and a significant reduction after MP only in corticosteroid-resistant GO patients. Soluble MMP-9 was highest in the GO group compared with both the Gd and control individuals. Moreover serum MMP-9 decreased in corticosteroid-responsive GO patients after MP and remained at the lower level at the end of the study. Positive correlations between MMP-2 and MMP-9 before and after MP administration were observed. Serum VCAM-1 was significantly elevated both in GO and Gd subjects and pre-treatment VCAM-1 levels were elevated in corticosteroid-responders compared with non-responders. CONCLUSIONS: Our results suggest that serum VCAM-1 may serve as a marker predicting the efficacy of corticosteroids and that soluble MMP-9 may be helpful in monitoring corticosteroid administration and in decision-making with regard to further GO treatment.     

Zinc supplementation inhibits lipid peroxidation and the development of atherosclerosis in rabbits fed a high cholesterol diet

Developing atherosclerotic lesions in hypercholesterolemic rabbits are depleted in zinc, while iron accumulates. This study examined the influence of zinc supplementation on the development of atherosclerosis and used isotope dilution gas chromatography-mass spectrometry techniques to measure biomarkers of oxidative lipid damage in atherosclerotic rabbit aorta. Our previous method for F(2)-isoprostane measurement was adapted to include the quantitation of cholesterol oxidation products in the same sample. Two groups of New Zealand white rabbits were fed a high cholesterol (1% w/w) diet and one group was also supplemented with zinc (1 g/kg) for 8 weeks. Controls were fed a normal diet. Zinc supplementation did not significantly alter the increase in total plasma cholesterol levels observed in animals fed high cholesterol. However, in cholesterol-fed animals zinc supplementation significantly reduced the accumulation of total cholesterol levels in aorta which was accompanied by a significant reduction in average aortic lesion cross-sectional areas of the animals. Elevated levels of cholesterol oxidation products (5,6-alpha and beta cholesterol epoxides, 7beta-hydroxycholesterol, 7-ketocholesterol) in aorta and total F(2)-isoprostanes in plasma and aorta of rabbits fed a cholesterol diet were significantly decreased by zinc supplementation. Our data indicate that zinc has an antiatherogenic effect, possibly due to a reduction in iron-catalyzed free radical reactions.     

Dietary flaxseed inhibits atherosclerosis in the LDL receptor-deficient mouse in part through antiproliferative and anti-inflammatory actions

Dietary flaxseed has been shown to have potent antiatherogenic effects in rabbits. The purpose of the present study was to investigate the antiatherogenic capacity of flaxseed in an animal model that more closely represents the human atherosclerotic condition, the LDL receptor-deficient mouse (LDLrKO), and to identify the cellular mechanisms for these effects. LDLrKO mice were administered a regular diet (RG), a 10% flaxseed-supplemented diet (FX), or an atherogenic diet containing 2% cholesterol alone (CH) or supplemented with 10% flaxseed (CF), 5% flaxseed (CF5), 1% flaxseed (CF1), or 5% coconut oil (CS) for 24 wk. LDLrKO mice fed a cholesterol-supplemented diet exhibited a rise in plasma cholesterol without a change in triglycerides and an increase in atherosclerotic plaque formation. The CS mice exhibited elevated levels of plasma cholesterol, triglycerides, and saturated fatty acids and an increase in plaque development. Supplementation of the cholesterol-enriched diet with 10% (wt/wt) ground flaxseed lowered plasma cholesterol and saturated fatty acids, increased plasma ALA, and inhibited plaque formation in the aorta and aortic sinus compared with mice fed a diet supplemented with only dietary cholesterol. The expression of proliferating cell nuclear antigen (PCNA) and the inflammatory markers IL-6, mac-3, and VCAM-1 was increased in aortic tissue from CH and CS mice. This expression was significantly reduced or normalized when flaxseed was included in the diet. Our results demonstrate that dietary flaxseed can inhibit atherosclerosis in the LDLrKO mouse through a reduction of circulating cholesterol levels and, at a cellular level, via antiproliferative and anti-inflammatory actions.     

Specific Matrix Metalloproteinases Play Different Roles in Intraplaque Angiogenesis and Plaque Instability in Rabbits

Background

Ectopic angiogenesis within the intima and media is considered to be a hallmark of advanced vulnerable atherosclerotic lesions. Some studies have shown that specific matrix metalloproteinases (MMPs) might play different roles in angiogenesis. Therefore, we investigated the predominant effects of specific MMPs in intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis.

Methods and Results

New Zealand rabbits underwent balloon injury of the abdominal artery and ingestion of a high-cholesterol (1%) diet to establish an atherosclerotic animal model. At weeks 4, 6, 8, 10, and 12 after balloon injury, five rabbits were euthanized and the abdominal aorta was harvested. Blood lipid analysis, intravascular ultrasound imaging, pathologic and immunohistochemical expression studies, and western blotting were performed. From weeks 4 to 12, the expression of MMP-1, -2, -3, and -9 and vascular endothelial growth factor A (VEGF-A) increased with atherosclerotic plaque development in the abdominal aorta, while the expression of MMP-14 substantially decreased. The vulnerability index (VI) gradually increased over time. Intraplaque neovessels appeared at week 8. The microvessel density (MVD) was greater at week 12 than at week 8. The VI, MVD, and VEGF-A level were positively correlated with the MMP-1, -2,-3, and -9 levels within plaques. Negative correlations were noted between the MMP-14 level and the VI, MVD, and VEGF-A level.

Conclusion

Upregulation of MMP-1, -2, -3, and -9 and downregulation of MMP-14 may contribute to intraplaque angiogenesis and plaque instability at the advanced stage of atherosclerosis in rabbits.     

Hazelnut oil administration reduces aortic cholesterol accumulation and lipid peroxides in the plasma, liver, and aorta of rabbits fed a high-cholesterol diet

Hazelnut oil (HO) is rich in monounsaturated fatty acids and antioxidants. We wanted to investigate the effect of HO on lipid levels and prooxidant-antioxidant status in rabbits fed a high-cholesterol (HC) diet. An HC diet caused significant increases in lipids and lipid peroxide levels in the plasma, liver, and aorta together with histopathological atherosclerotic changes in the aorta. Glutathione levels, glutathione peroxidase, and glutathione transferase activities decreased significantly, but superoxide dismutase activity and vitamin E and C levels remained unchanged in the livers of rabbits following HC diet. HO supplementation reduced plasma, liver, and aorta lipid peroxide levels and aorta cholesterol levels together with amelioration in atherosclerotic lesions in the aortas of rabbits fed an HC diet, without any decreasing effect on cholesterol levels in the plasma or liver. HO did not alter the antioxidant system in the liver in the HC group. Our findings indicate that HO reduced oxidative stress and cholesterol accumulation in the aortas of rabbits fed an HC diet.     

IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2('),7(')-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.     

Nicotine promotes atherosclerosis development in apolipoprotein E-deficient mice through α1-nAChR

α1 Nicotinic acetylcholine receptor (α1nAChR) is an important nicotine receptor that is widely distributed in vascular smooth muscle cells, macrophages, and endothelial cells. However, the role of α1nAChR in nicotine-mediated atherosclerosis remains unclear. The administration of nicotine for 12 weeks increased the area of the atherosclerotic lesion, the number of macrophages infiltrating the plaques, and the circulating levels of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α, in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet. Nicotine also increased α1nAChR, calpain-1, matrix metalloproteinase-2 (MMP-2), and MMP-9 expression in the aortic tissue. Silencing of α1nAChR with an adenoassociated virus decreased the atherosclerotic size, lesion macrophage content, and circulating levels of inflammatory cytokines and suppressed α1nAChR, calpain-1, MMP-2, and MMP-9 expression in the nicotine group. In vitro, nicotine-induced α1nAChR, calpain-1, MMP-2, and MMP-9 expression in mouse vascular smooth muscle cells (MOVAS) and macrophages (RAW264.7), and enhanced the migration and proliferation of these cells. The silencing of α1nAChR inhibited these effects of nicotine MOVAS and RAW264.7 cells. Thus, we concluded that nicotine promoted the development of atherosclerosis partially by inducing the migration and proliferation of vascular smooth muscle cells and macrophages and inducing an inflammatory reaction. The effect of nicotine on atherogenesis may be mediated by α1nAChR-induced activation of the calpain-1/MMP-2/MMP-9 signaling pathway.     

Osteopontin inhibits interleukin-1beta-stimulated increases in matrix metalloproteinase activity in adult rat cardiac fibroblasts: role of protein kinase C-zeta

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1beta (IL-1beta), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1beta-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycine-arginine-glycine-aspartic acid-serine)-pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat beta3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1beta increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1beta-stimulated increases in translocation of PKC-zeta from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-zeta were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-zeta using PKC-zeta pseudosubstrate inhibited IL-1beta-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via beta3 integrins, inhibits IL-1beta-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-zeta. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity.     

Macrophage-derived MMP-9 enhances the progression of atherosclerotic lesions and vascular calcification in transgenic rabbits

Matrix metalloproteinase-9 (MMP-9), or gelatinase B, has been hypothesized to be involved in the progression of atherosclerosis. In the arterial wall, accumulated macrophages secrete considerable amounts of MMP-9 but its pathophysiological functions in atherosclerosis have not been fully elucidated. To examine the hypothesis that macrophage-derived MMP-9 may affect atherosclerosis, we created MMP-9 transgenic (Tg) rabbits to overexpress the rabbit MMP-9 gene under the control of the scavenger receptor A enhancer/promoter and examined their susceptibility to cholesterol diet-induced atherosclerosis. Tg rabbits along with non-Tg rabbits were fed a cholesterol diet for 16 and 28 weeks, and their aortic and coronary atherosclerosis was compared. Gross aortic lesion areas were significantly increased in female Tg rabbits at 28 weeks; however, pathological examination revealed that all the lesions of Tg rabbits fed a cholesterol diet for either 16 or 28 weeks were characterized by increased monocyte/macrophage accumulation and prominent lipid core formation compared with those of non-Tg rabbits. Macrophages isolated from Tg rabbits exhibited higher infiltrative activity towards a chemoattractant, MCP-1 in vitro and augmented capability of hydrolysing extracellular matrix in granulomatous tissue. Surprisingly, the lesions of Tg rabbits showed more advanced lesions with remarkable calcification in both aortas and coronary arteries. In conclusion, macrophage-derived MMP-9 facilitates the infiltration of monocyte/macrophages into the lesions thereby enhancing the progression of atherosclerosis. Increased accumulation of lesional macrophages may promote vascular calcification.     

Inhibition of lipoprotein-associated phospholipase A2 ameliorates inflammation and decreases atherosclerotic plaque formation in ApoE-deficient mice

Background

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is thought to play modulatory roles in the development of atherosclerosis. Here we evaluated the effects of a specific lp-PLA2 inhibitor on atherosclerosis in ApoE-deficient mice and its associated mechanisms.

Methodology/Principal Findings

ApoE-deficient mice fed an atherogenic high-fat diet for 17 weeks were divided into two groups. One group was administered the specific lp-PLA2 inhibitor, darapladib (50 mg/kg/day; p.o.) daily for 6 weeks, while the control group was administered saline. We observed no differences in body weight and serum lipids levels between the two groups at the end of the dietary period. Notably, serum lp-PLA2 activity as well as hs-CRP (C-reactive protein) and IL-6 (Interleukin-6) levels were significantly reduced in the darapladib group, compared with the vehicle group, while the serum PAF (platelet-activating factor) levels were similar between the two groups. Furthermore, the plaque area through the arch to the abdominal aorta was reduced in the darapladib group. Another finding of interest was that the macrophage content was decreased while collagen content was increased in atherosclerotic lesions at the aortic sinus in the darapladib group, compared with the vehicle group. Finally, quantitative RT-PCR performed to determine the expression patterns of specific inflammatory genes at atherosclerotic aortas revealed lower expression of MCP-1, VCAM-1 and TNF-α in the darapladib group.

Conclusions/Significance

Inhibition of lp-PLA2 by darapladib leads to attenuation of in vivo inflammation and decreased plaque formation in ApoE-deficient mice, supporting an anti-atherogenic role during the progression of atherosclerosis.     

Citreoviridin Enhances Atherogenesis in Hypercholesterolemic ApoE-Deficient Mice via Upregulating Inflammation and Endothelial Dysfunction

Vascular endothelial dysfunction and inflammatory response are early events during initiation and progression of atherosclerosis. In vitro studies have described that CIT markedly upregulates expressions of ICAM-1 and VCAM-1 of endothelial cells, which result from NF-κB activation induced by CIT. In order to determine whether it plays a role in atherogenesis in vivo, we conducted the study to investigate the effects of CIT on atherosclerotic plaque development and inflammatory response in apolipoprotein E deficient (apoE^-/-) mice. Five-week-old apoE^-/- mice were fed high-fat diets and treated with CIT for 15 weeks, followed by assay of atherosclerotic lesions. Nitric oxide (NO), vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were detected in serum. Levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), VEGF, and ET-1 in plaque areas of artery walls were examined. NF-κB p65 expression and NF-κB activation in aorta also were assessed. CIT treatment significantly augmented atherosclerotic plaques and increased expressions of ICAM-1, VCAM-1, VEGF and ET-1 in aorta. Mechanistic studies showed that activation of NF-κB was significantly elevated by CIT treatment, indicating the effect of CIT on atherosclerosis may be regulated by activation of NF-κB.     

A cis-9,trans-11-conjugated linoleic acid-rich oil reduces the outcome of atherogenic process in hyperlipidemic hamster

Conjugated linoleic acid (CLA) mixtures demonstrated antiatherogenic properties in several animal models, including hamsters, but the mechanism of action of the main food-derived CLA isomer is unknown in this species. This study thus focused on cis-9,trans-11-CLA (rumenic acid), and its effect was compared with that of fish oil, which is known to influence several aspects of atherogenesis. Syrian hamsters were fed (for 12 wk) diets containing 20% (wt/wt) butter fat (B diet) or the same diet augmented with either 1% (wt/wt) of a cis-9,trans-11-CLA-rich oil (BR diet) or 1% (wt/wt) fish oil (BF diet). The BR diet induced the lowest aortic lipid deposition (from -30% to -45%) among the butter oil-fed hamsters. In this group, plasma also displayed a reduced non-HDL-to-HDL-cholesterol ratio (21% less than in the butter oil group) and inflammatory serum amyloid A levels (70-80%) and an improvement of anti-oxidized LDL paraoxonase activity (all P < 0.05). Compared with the B group, the beneficial effects of the BR diet could be further explained in part by preventing the high VCAM-1 expression rate, increasing (30%) ATP-binding cassette subfamily A1 expression in the aorta, and downregulating expression of inflammatory-related genes (TNF-alpha, IL-1beta, and cyclooxygenase 2, 2- to 2.8-fold, P < 0.05). This effect was partly associated with an activation of peroxisome proliferator-activating receptor (PPAR)/liver X receptor (LXR)-alpha signaling cascade. Interestingly, activation of PPAR/LXR-alpha signaling was not observed in hamsters fed the BF diet, in which the early signs of atherogenesis were increased. In conclusion, this study demonstrated that milk fat-rich cis-9,trans-11-CLA reduces the atherogenic process in hyperlipidemic hamsters.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

In vitro PGI2 synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI2 produced were shown as 6-keto-PGF1 alpha per microgram of aortic tissue DNA instead of per mg wet weight. We also investigated PGI2 synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima. Basal PGI2 production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI2 production by atherosclerotic aorta was also significantly higher than that of controls. PGI2 producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI2 production than the medial layer. Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI2 than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI2 production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Matrix metalloproteinase (MMP)-12 regulates MMP-9 expression in interleukin-1beta-treated articular chondrocytes

Limited information is available on the expression and role of matrix metalloproteinase (MMP)-12 in chondrocytes. We characterized the expression mechanism of MMP-12 and possible function in chondrocytes. Interleukin (IL)-1beta induced the expression and activation of MMP-12 in primary culture chondrocytes and cartilage explants via mitogen-activated protein (MAP) kinase signaling pathways. Among MAP kinases, extracellular signal-regulated kinase and p38 kinase are necessary for MMP-12 expression, whereas c-jun N-terminal kinase is required for the activation of MMP-12. The possibility that MMP-12 acts as a modulator of other MMP was examined. MMP-12 alone did not affect other MMP expressions. However, MMP-12 enhanced expression and activation of MMP-9 in the presence of IL-1beta. Our results indicate that IL-1beta in chondrocytes induces the expression and activation of MMP-12, which, in turn, augments MMP-9 expression and activation.