Functional alterations in endothelial NO, PGI₂ and EDHF pathways in aorta in ApoE/LDLR⁻/⁻ mice

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI?) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI?-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR?/? mice. We found that in the aorta of 2-month-old apoE/LDLR?/? mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI? and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR?/? mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI? and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI? and EDHF pathways may compensate for the loss of the biological activity of NO.     

apoE/LDLR双基因缺失幼龄小鼠主动脉中动脉粥样硬化相关基因的表达

利用RT-PCR以及实时定量RT-PCR检测11个动脉粥样硬化(atherosclerosis,AS)相关基因在1、2和3月龄的载脂蛋白E(apolipoproteinE,aopE)/低密度脂蛋白受体(low-density lipoprotein receptor,LDLR)双基因缺失(apoE-/-/LDLR-/-)小鼠主动脉中的表达变化,同时应用血生化指标和病理形态学观察AS早期病变特点,探讨apoE和LDLR基因联合缺失引发的血脂代谢紊乱和血管炎症损伤的关系以及AS的炎症反应机制.结果显示,apoE-/-/LDLR-/-小鼠IL-18、TLR2、MCP-1、ICAM-1、VCAM-1、GM-CSF、CD36和ET-1表达在1月龄时较同龄野生型(wild type,WT)小鼠显著上调(P<0.05,P<0.01),PDGF-α和TNF-α表达在2月龄时较同龄WT小鼠显著上调,除ET-1表达在2月龄时以及了LR2、VCAM-1和ICAM-1表达在3月龄时降至WT小鼠水平以外,其余各基因表达随年龄增长继续升高(P<0.05,P<0.01),其中MCP-1表达在2月龄时达到峰值.NF-kB在各年龄段apoE-/-/LDLR-/-小鼠中的表达与同龄WT小鼠相比均无显著差异.各年龄段apoE-/-/LDLR-/-/小鼠血清了C、TG、LDL、HDL、TNF-α、IL-1β和ox-LDL含量均显著高于同龄WT小鼠(P<0.05,P<0.01),并随年龄增长逐渐升高.apoE-/-/LDLR-/-小鼠1月龄时主动脉内膜出现少量的散在的脂质沉积,随着年龄增长病变区域增多,脂质沉积增厚.上述结果提示:apoE和LDLR双基因缺失形成的高脂血症可能通过刺激主动脉中炎症基因时序表达,起始并扩大病变部位的炎症反应,共同促进AS的发生发展.     

Triple immunofluorescence labeling of atherosclerotic plaque components in apoE/LDLR -/- mice

This paper presents a simple and reliable method of triple immunofluorescence staining that allows simultaneous detection of various cell types present in atherosclerotic plaque of apolipoprotein E and LDL receptor-double knockout (apoE/LDLR -/-) mice. We used combined direct and indirect procedures applying commercially available primary antibodies raised in different species to detect smooth muscle cells (Cy3-conjugated mouse anti-smooth muscle actin, SMA), macrophages (rat anti-CD68) and T lymphocytes (rabbit anti-CD3). Fixation of the material in acetone and modified incubation protocol employing nonfat dry milk in preincubation and incubation media significantly increased the intensity of labeling and effectively quenched the background. Our method offers an efficient way to detect qualitative as well as quantitative changes of macrophages, T lymphocytes and smooth muscle cells in atherosclerotic plaque of apoE/LDLR -/- mice during atherosclerosis development or in response to pharmacological treatment.     

Effect of macrophage-derived apolipoprotein E on hyperlipidemia and atherosclerosis of LDLR-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice fed a Western diet exhibit severe hyperlipidemia and develop significant atherosclerosis. Apolipoprotein E (apoE) is a multifunctional protein synthesized by hepatocytes and macrophages. We sought to determine effect of macrophage apoE deficiency on severe hyperlipidemia and atherosclerosis. Female LDLR(-/-) mice were lethally irradiated and reconstituted with bone marrow from either apoE(-/-) or apoE(+/+) mice. Four weeks after transplantation, recipient mice were fed a Western diet for 8 weeks. Reconstitution of LDLR(-/-) mice with apoE(-/-) bone marrow resulted in a slight reduction in plasma apoE levels and a dramatic reduction in accumulation of apoE and apoB in the aortic wall. Plasma lipid levels were unaffected when mice had mild hyperlipidemia on a chow diet, whereas IDL/LDL cholesterol levels were significantly reduced when mice developed severe hyperlipidemia on the Western diet. The hepatic VLDL production rate of mice on the Western diet was decreased by 46% as determined by injection of Triton WR1339 to block VLDL clearance. Atherosclerotic lesions in the proximal aorta were significantly reduced, partially due to reduction in plasma total cholesterol levels (r=0.56; P<0.0001). Thus, macrophage apoE-deficiency alleviates severe hyperlipidemia by slowing hepatic VLDL production and consequently reduces atherosclerosis in LDLR(-/-) mice.     

Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein E

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivo oxidant stress, we quantitated the isoprostane iPF(2 alpha)-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF(2 alpha)-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.     

Characterization of Ath29, a major mouse atherosclerosis susceptibility locus, and identification of Rcn2 as a novel regulator of cytokine expression

Ath29 is an atherosclerosis susceptibility locus on chromosome 9 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE(-/-)) mice. This locus was subsequently replicated in two separate intercrosses that developed early or advanced atherosclerotic lesions. The objective of this study was to characterize Ath29 through construction and analysis of a congenic strain and identify underlying candidate genes. A congenic line was constructed by introgressing the chromosomal segment harboring Ath29 from C3H.apoE(-/-) into B6.apoE(-/-) mice. Congenic mice developed significantly smaller early and advance atherosclerotic lesions than B6.apoE(-/-) mice. Microarray analysis revealed 317 genes to be differentially expressed in the aorta of congenic mice compared with B6.apoE(-/-) mice. Pathway analysis of these genes suggested the Ca(2+) signaling pathway to be implicated in regulating atherosclerosis susceptibility. Rcn2 is located underneath the linkage peak of Ath29 and involved in Ca(2+) signaling. Multiple single-nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Rcn2 with one single-nucleotide polymorphism falling within an upstream cAMP response element. Immunostaining demonstrated its expression in atherosclerotic lesions. Knockdown of Rcn2 with small interfering RNAs resulted in significant reductions in both baseline and oxidized phospholipid-induced VCAM-1 and monocyte chemoattractant protein-1 expression by endothelial cells. Ath29 is confirmed to be a major atherosclerosis susceptibility locus affecting both early and advanced lesion formation in mice, and Rcn2 is identified as a novel regulator of cytokine expression.     

Suppression of atherogenesis by delivery of TGFbeta1ACT using adeno-associated virus type 2 in LDLR knockout mice

TGFbeta(1) deficiency has been attributed to the development of atherosclerosis. There is, however, little direct evidence for this concept. To examine this hypothesis, low-density lipoprotein receptor knockout (LDLR(-/-)) mice were injected via tail vein with recombinant adeno-associated virus type 2 (rAAV) carrying a bioactive TGFbeta(1) mutant (AAV/TGFbeta1ACT, n=10) or granulocyte-macrophage-colony stimulating factor (AAV/GM-CSF, n=10, a negative control) or saline (n=9, control), and then put on a high cholesterol diet. At 18 weeks, blood lipids were found to be similarly elevated in all LDLR(-/-) mice. TGFbeta1ACT and GM-CSF (DNA, mRNA, and protein) were highly expressed in the tissues of mice given TGFbeta1ACT or AAV/GM-CSF, respectively, showing sustained transfection following gene delivery by the systemic route. Saline-treated and AAV/GM-CSF-treated LDLR(-/-) mice showed extensive areas of atherosclerotic lesion formation. There was evidence of intense oxidative stress (nitrotyrosine staining), inflammation (CD68 staining), and expression of adhesion molecules and the ox-LDL receptor LOX-1 (gene array analysis) in the atherosclerotic tissues. Importantly, atherosclerotic lesion formation was markedly inhibited in the LDLR(-/-) mice given AAV/TGFbeta1ACT. Expression of adhesion molecules and LOX-1, oxidative stress, and inflammatory response all were inhibited in the mice given AAV/TGFbeta1ACT (P<0.05 vs. saline-treated or GM-CSF-treated LDLR(-/-) mice). These data for the first time demonstrate that systemic delivery of TGFbeta1ACT gene via AAV can inhibit formation of atherosclerotic lesions, possibly via anti-inflammatory and anti-oxidant mechanisms. These findings suggest a novel view of TGFbeta(1) in atherogenesis and a potential new gene therapy for treatment of atherosclerosis.     

Manzamine A,a marine-derived alkaloid,inhibits accumulation of cholesterol ester in macrophages and suppresses hyperlipidemia and atherosclerosis in vivo

The formation of foam cells in macrophages plays an essential role in the progression of early atherosclerotic lesions and therefore its prevention is considered to be a promising target for the treatment of atherosclerosis. We found that an extract of the marine sponge Acanthostrongylophora ingens inhibited the foam cell formation induced by acetylated low-density lipoprotein (AcLDL) in human monocyte-derived macrophages, as measured based on the accumulation of cholesterol ester (CE). Bioassay-guided purification of inhibitors from the extract afforded manzamines. Manzamine A was the most potent inhibitor of foam cell formation, and also suppressed CE formation in Chinese hamster ovary cells overexpressing acyl-CoA:cholesterol acyl-transferase (ACAT)-1 or ACAT-2. In addition, manzamine A inhibited ACAT activity. Next, we orally administered manzamine A to apolipoprotein E (apoE)-deficient mice for 80 days, and found that total cholesterol, free cholesterol, LDL-cholesterol, and triglyceride levels in serum were significantly reduced and the area of atherosclerotic lesions in the aortic sinus was also substantially diminished. These findings clearly suggest that manzamine A suppresses hyperlipidemia and atherosclerosis in apoE-deficient mice by inhibiting ACAT and is therefore a promising lead compound in the prevention or treatment of atherosclerosis. Although manzamine A has been reported to show several biological activities, this is the first report of a suppressive effect of manzamine A on atherosclerosis in vivo.     

Defective VLDL metabolism and severe atherosclerosis in mice expressing human apolipoprotein E isoforms but lacking the LDL receptor

Differences in affinity of human apolipoprotein E (apoE) isoforms for the low density lipoprotein receptor (LDLR) are thought to result in the differences in lipid metabolism observed in humans with different APOE genotypes. Mice expressing three common human apoE isoforms, E2, E3, and E4, in place of endogenous mouse apoE were used to investigate the relative roles of apoE isoforms in LDLR- and non-LDLR-mediated very low density lipoprotein (VLDL) clearance. While both VLDL particles isolated from mice expressing apoE3 and apoE4 bound to mouse LDLR with affinity and Bmax similar to VLDL containing mouse apoE, VLDL with apoE2 bound with only half the Bmax. In the absence of the LDLR, all lines of mice expressing human apoE showed dramatic increases in VLDL cholesterol and triglycerides (TG) compared to LDLR knockout mice expressing mouse apoE. The mechanism of the hyperlipidemia in mice expressing human apoE isoforms is due to impairment of non-LDL-receptor-mediated VLDL clearance. This results in the severe atherosclerosis observed in mice expressing human apoE but lacking the LDLR, even when fed normal chow diet. Our data show that defects in LDLR independent pathway(s) are a potential factor that trigger hyperlipoproteinemia when the LDLR pathway is perturbed, as in E2/2 mice.     

Attenuated atherosclerotic lesions in apoE-Fcγ-chain-deficient hyperlipidemic mouse model is associated with inhibition of Th17 cells and promotion of regulatory T cells

Though the presence of antioxidized low-density lipoprotein IgG is well documented in clinical and animal studies, the role for FcγRs to the progression of atherosclerosis has not been studied in detail. In the current study, we investigated the role for activating FcγR in the progression of atherosclerosis using apolipoprotein E (apoE)-Fcγ-chain double-knockout (DKO) mice. Relative to apoE knockout (KO) mice, arterial lesion formation was significantly decreased in apoE-Fcγ-chain DKO mice. Bone marrow chimera studies showed reduced lesions in apoE KO mice receiving the bone marrow of apoE-Fcγ-chain DKO mice. Compared to apoE KO mice, antioxidized low-density lipoprotein IgG1 (Th2) and IgG2a (Th1), IL-10, and IFN-γ secretion by activated T cells was increased in apoE-Fcγ-chain DKO mice. These findings suggest that reduced atherosclerotic lesion in apoE-Fcγ-chain DKO mice is not due to a Th1/Th2 imbalance. Interestingly, the number of Th17 cells and the secretion of IL-17 by activated CD4(+) cells were decreased in apoE-Fcγ-chain DKO mice. Notably, the number of regulatory T cells, expression of mRNA, and secretion of TGF-β and IL-10 were increased in apoE-Fcγ-chain DKO mice. Furthermore, secretions of IL-6 and STAT-3 phosphorylation essential for Th17 cell genesis were reduced in apoE-Fcγ-chain DKO mice. Importantly, decrease in Th17 cells in apoE-Fcγ-chain DKO mice was due to reduced IL-6 release by APC of apoE-Fcγ-chain DKO mice. Collectively, our data suggest that activating FcγR promotes atherosclerosis by inducing a Th17 response in the hyperlipidemic apoE KO mouse model.     

Mouse models of experimental atherosclerosis.

Since 1992 the mouse has become an excellent model for experimental atherosclerosis research. Until 1992, the diet -- induced atherosclerosis mouse model has been used effectively, but the lesions tended to be small and were limited to early fatty-streak stage. This model was also criticized because of the toxicity and inflammatory responses due to the diet. In 1992 the first line of gene targeted animal models, namely apolipoprotein E -- knockout mice was developed. Of the genetically engineered models, the apoE -- deficient model is the only one that develops extensive atherosclerotic lesions on a chow diet. It is also the model in which the lesions have been characterized most thoroughly. The lesions develop into fibrous plaques; however, there is no evidence that plaque rupture occurs in this model. The LDL receptor - deficient model has elevated LDL levels, but no lesions, or only very small lesions, form on the chow diet, however, robust lesions do form on the western-type diet. The creation of apoE -- knockout mice has changed the face of atherosclerosis research.     

Endothelin-converting enzyme-1 increases in atherosclerotic mice: potential role of oxidized low density lipoproteins

The aim of our study was to analyze the relationships between atherosclerosis and endothelin-converting enzyme-1 (ECE-1). Four-week-old C57BL/6J [wild-type (WT)] and apolipoprotein E-deficient (apoE) mice were fed with a standard or Western-type fat diet for 8 wks. ApoE showed atherosclerotic lesions in the aorta, higher blood pressure and vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein content than WT. ApoE showed a significant increase in ECE-1 protein content and mRNA expression in aorta, lung, and kidney, without changes in heart. When an ECE-1 inhibitor, FR-901533, was administered to them, blood pressure decreased in apoE on fat diet versus apoE on normal diet and WT. ECE-1 and LOX-1 protein content were elevated in peripheral blood mononuclear cells (PBMC) from hypercholesterolemic patients. In order to study the mechanism involved in this ECE-1 up-regulation, bovine aortic endothelial cells (BAEC) were treated with oxidized-low density lipoproteins (oxLDL). OxLDL, but not LDL, increased ECE-1 protein content, mRNA expression and promoter activity. Our results demonstrate that ECE-1 increases in different atherosclerosis situations. Up-regulation of ECE-1 could contribute, at least partially, to the development of hypertension seen in apoE mice, because FR-901533 avoided it. Probably, atherosclerotic situations course with an increase of oxLDL, which is able to induce ECE-1 expression with the subsequent potential pathological effects.     

Atherosclerosis in perlecan heterozygous mice

The hypothesis that lipoprotein association with perlecan is atherogenic was tested by studying atherosclerosis in mice that had a heterozygous deletion of perlecan, the primary extracellular heparan sulfate proteoglycan in arteries. We first studied the expression of perlecan in mouse lesions and noted that this proteoglycan in aorta was found in the subendothelial matrix. Perlecan was also a major component of the lesional extracellular matrix. Mice with a heterozygous deletion had a reduction in arterial wall perlecan expression. Atherosclerosis in these mice was studied after crossing the defect into the apolipoprotein E (apoE) and LDL receptor knockout backgrounds. At 12 weeks, chow-fed apoE null mice with a heterozygous deletion had less atherosclerosis. However, at 24 weeks and in the LDL receptor heterozygous background, the presence of a perlecan knockout allele did not significantly alter lesion size. Thus, it appears that loss of perlecan leads to less atherosclerosis in early lesions. Although this might be attributable to a decrease in lipoprotein retention, it should be noted that perlecan might mediate multiple other processes that could, in sum, accelerate atherosclerosis.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

The role of hepatic Surf4 in lipoprotein metabolism and the development of atherosclerosis in apoE−/− mice

Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr^?/?) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE^?/?) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE^?/? mice. In apoE^?/? mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE^?/? mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.     

Physiological expression of macrophage apoE in the artery wall reduces atherosclerosis in severely hyperlipidemic mice

We have previously reported that the introduction of macrophage apoE into mice lacking both apoE and the LDL receptor (apoE(-)(/-)/LDLR(-)(/-)) through bone marrow transplantation (apoE(+)(/+)/LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-)) produces progressive accumulation of apoE in plasma without affecting lipid levels. This model provides a tool to study the effects of physiologically regulated amounts of macrophage apoE on atherogenesis in hyperlipidemic animals. Ten-week-old male apoE(-)(/-)/LDLR(-)(/-) mice were transplanted with either apoE(+)(/+)/LDLR(-)(/-) (n = 11) or apoE(-)(/-)/LDLR(-)(/-) (n = 14) marrow. Although there were no differences between the two groups in lipid levels at baseline or at 5 and 9 weeks after transplantation, apoE levels in the apoE(+)(/+)LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-) mice increased to 4 times the apoE levels of normal mice. This resulted in a 60% decrease in aortic atherosclerosis in the apoE(+)(/+)/LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-) compared with the apoE(-)(/-)/LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-) controls, (15957 +/- 1907 vs. 40115 +/- 8302 micro m(2) +/- SEM, respectively). In a separate experiment, apoE(+)(/+)/LDLR(-)(/-) mice were transplanted with either apoE(+)(/+)/LDLR(-)(/-) or apoE(-)(/-)/LDLR(-)(/-) marrow and placed on a high-fat diet for 8 weeks. In the absence of macrophage apoE, lesion area was increased by 75% in the aortic sinus and by 56% in the distal aorta. These data show that physiologic levels of macrophage apoE in the vessel wall are anti-atherogenic in conditions of severe hyperlipidemia and can affect later stages of plaque development.     

Dietary sodium chloride restriction enhances aortic wall lipid storage and raises plasma lipid concentration in LDL receptor knockout mice

This study aimed at measuring the influence of a low salt diet on the development of experimental atherosclerosis in moderately hyperlipidemic mice. Experiments were carried out on LDL receptor (LDLR) knockout (KO) mice, or apolipoprotein E (apoE) KO mice on a low sodium chloride diet (LSD) as compared with a normal salt diet (NSD). On LSD, the rise of the plasma concentrations of TG and nonesterified fatty acid (NEFA) was, respectively, 19% and 34% in LDLR KO mice, and 21% and 35% in apoE KO mice, and that of plasma cholesterol was limited to the LDLR KO group alone (15%). Probably due to the apoE KO severe hypercholesterolemia, the arterial inner-wall fat storage was not influenced by the diet salt content and was far more abundant in the apoE KO than in the LDLR KO mice. However, in the less severe hypercholesterolemia of the LDLR KO mice, lipid deposits on the LSD were greater than on the NSD. Arterial fat storage correlated with NEFA concentrations in the LDLR KO mice alone (n = 14, P = 0.0065). Thus, dietary sodium chloride restriction enhances aortic wall lipid storage in moderately hyperlipidemic mice.     

Circulating adhesion molecules in apoE-deficient mouse strains with different atherosclerosis susceptibility

Recruitment of inflammatory cells in the arterial wall by vascular adhesion molecules plays a key role in development of atherosclerosis. Apolipoprotein E-deficient (apoE(-/-)) mice have spontaneous hyperlipidemia and develop all phases of atherosclerotic lesions. We sought to examine plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and sP-selectin in two apoE(-/-) strains C57BL/6 (B6) and BALB/c with early or advanced lesions. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. On either diet, BALB/c.apoE(-/-) mice developed much smaller atherosclerotic lesions and displayed significantly lower levels of sVCAM-1 and sP-selectin than B6.apoE(-/-) mice. The Western diet significantly elevated sVCAM-1 levels in both strains and sP-selectin levels in B6.apoE(-/-) mice. BALB/c.apoE(-/-) mice exhibited 2-fold higher HDL cholesterol levels on the chow diet and 15-fold higher HDL levels on the Western diet than B6.apoE(-/-) mice, although the two strains had comparable levels of total cholesterol and triglyceride. Thus, increased atherosclerosis is accompanied by increases in circulating VCAM-1 and P-selectin levels in the two apoE(-/-) mouse strains, and the high HDL level may protect against atherosclerosis by inhibiting the expression of adhesion molecules in BALB/c.apoE(-/-) mice.     

Protection of atherogenesis in thromboxane A2 receptor-deficient mice is not associated with thromboxane A2 receptor in bone marrow-derived cells

In the previous study, we generated mice lacking thromboxane A2 receptor (TP) and apolipoprotein E, apoE(-/-)TP(-/-) mice, and reported that the double knockout mice developed markedly smaller atherosclerotic lesions than those in apoE(-/-) mice. To investigate the mechanism responsible for reduced atherosclerosis in apoE(-/-)TP(-/-) mice, we examined the role of TP in bone marrow (BM)-derived cells in the development of the atherosclerotic lesions. When we compared the function of macrophages in apoE(-/-) and in apoE(-/-)TP(-/-) mouse in vitro, there was no difference in the expression levels of cytokines and chemokines after stimulation with lipopolysaccharide. We then transplanted the BM from either apoE(-/-) or apoE(-/-)TP(-/-) mice to either apoE(-/-) or apoE(-/-)TP(-/-) mice after sublethal irradiation. After 12 weeks with high fat diet, we analyzed the atherosclerotic lesion of aortic sinus. When the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-) mice, the lesion size was almost the same as that of apoE(-/-) mice without BM transplantation. In contrast, when the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-)TP(-/-) mice, the lesion size was markedly reduced. These results indicate that the protection of atherogenesis in TP(-/-) mice is not associated with TP in BM-derived cells.