Atherosclerosis In Apolipoprotein E-knockout Mice Research Articles

High-fat-diet-induced obesity promotes simultaneous progression of lung cancer and atherosclerosis in apolipoprotein E-knockout mice.

Clinical studies have shown that atherosclerotic cardiovascular disease and cancer often co-exist in the same individual. The present study aimed to investigate the role of high-fat-diet (HFD)-induced obesity in the coexistence of the two diseases and the underlying mechanism in apolipoprotein E-knockout (ApoE-/-) mice. Male ApoE-/- mice were fed with a HFD or a normal diet (ND) for 15 weeks. On the first day of Week 13, the mice were inoculated subcutaneously in the right axilla with Lewis lung cancer cells. At Weeks 12 and 15, serumlectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and vascular endothelial growth factor levels were measured by enzyme-linked immunosorbent assay, and blood monocytes and macrophages were measured by fluorescence-activated cell sorting. At Week 15, the volume and weight of the local subcutaneous lung cancer and metastatic lung cancer and the amount of aortic atherosclerosis were measured. At Week 15, compared with mice in the ND group, those in the HFD group had a larger volume of local subcutaneous cancer (p = 0.0004), heavier tumors (p = 0.0235), more metastatic cancer in the lungs (p < 0.0001), a larger area of lung involved in metastatic cancer (p = 0.0031), and larger areas of atherosclerosis in the aorta (p < 0.0001). At Week 12, serum LOX-1, serum vascular endothelial growth factor, and proportions of blood monocytes and macrophages were significantly higher in the HFD group than those in the ND group (p = 0.0002, p = 0.0029, p = 0.0480, and p = 0.0106, respectively); this trend persisted until Week 15 (p = 0.0014, p = 0.0012, p = 0.0001, and p = 0.0204). In this study, HFD-induced obesity could simultaneously promote progression of lung cancer and atherosclerosis in the same mouse. HFD-induced upregulation of LOX-1 may play an important role in the simultaneous progression of these two conditions via the inflammatory response and VEGF.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice.

Atherosclerosis (AS), a chronic inflammatory disease of blood vessels, is a major contributor to cardiovascular disease. Dental pulp stem cells (DPSCs) are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflammation-related diseases. Hepatocyte growth factor (HGF) is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases. To modify DPSCs with HGF (DPSC-HGF) and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout (ApoE-/-) mouse model and an in vitro cellular model. ApoE-/- mice were fed with a high-fat diet (HFD) for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs (DPSC-Null) through tail vein at weeks 4, 7, and 11, respectively, and the therapeutic efficacy and mechanisms were analyzed by histopathology, flow cytometry, lipid and glucose measurements, real-time reverse transcription polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay at the different time points of the experiment. An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells (HAOECs), and indirect co-cultured with supernatant of DPSC-Null (DPSC-Null-CM) or DPSC-HGF-CM, and the effect and mechanisms were analyzed by flow cytometry, RT-PCR and western blot. Nuclear factor-κB (NF-κB) activators and inhibitors were also used to validate the related signaling pathways. DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors, and the percentage of macrophages in the aorta, and DPSC-HGF treatment had more pronounced effects. DPSCs treatment had no effect on serum lipoprotein levels. The FACS results showed that DPSCs treatment reduced the percentages of monocytes, neutrophils, and M1 macrophages in the peripheral blood and spleen. DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-α stimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway. This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/- mice on a HFD, and could be of greater value in stem cell-based treatments for AS.

Sinomenine protects against atherosclerosis in apolipoprotein E-knockout mice by inhibiting of inflammatory pathway.

Atherosclerosis, a multifaceted and persistent inflammatory condition, significantly contributes to the progression of cardiocerebrovascular disorders, such as myocardial infarctions and cerebrovascular accidents. It involves the accumulation of cholesterol, fatty deposits, calcium and cellular debris in the walls of arteries, leading to the formation of plaques. Our aim is to investigate the potential of sinomenine to counteract atherosclerosis in mice lacking Apolipoprotein E (ApoE-/-) Mice. We employed the high-fat diet-induced method to induce atherosclerosis in ApoE-/- mice, and the mice were treated with sinomenine (5, 10, and 15mg/kg) and simvastatin (0.5mg/kg) for 12weeks. Body weight, water intake, and food intake were assessed. Lipid parameters, oxidative stress, inflammatory cytokines, and mRNA levels were estimated. Sinomenine treatment remarkably (P < 0.001) suppressed body weight, along with food and water intake. Sinomenine altered the levels of total cholesterol (TC), high-density lipoprotein (HDL), triglyceride (TG), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), which were modulated in the atherosclerosis group. Sinomenine treatment also altered the levels of oxidative stress parameters such as glutathione peroxidase (GPx), catalase (CAT), malonaldehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH). In addition, it modulated cardiac parameters like C-reactive protein (CRP), endothelin-1 (ET-1), thromboxane B2 (TXB2), nitric oxide (NO), cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatinine kinase isoenzymes (CK-MB). Inflammatory cytokines interleukin (IL)-1α, IL-1β, TNF-α, IL-6, and IL-10 were also affected. Sinomenine further suppressed the mRNA expression of IL-6, IL-17, IL-10, tumor necrosis factor-α (TNF-α), Il-1β, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, transforming Growth Factor-1β (TGF-1β), vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). The results suggest that sinomenine remarkably suppressed the development of atherosclerosis in the early stage.

Time-restricted feeding prevents high-fat and high-cholesterol diet-induced obesity but fails to ameliorate atherosclerosis in apolipoprotein E-knockout mice.

One of the leading risk factors for atherosclerosis is obesity, which is commonly caused by a nutrient-rich Western-style diet, sedentary behaviors, and shift work. Time-restricted (TR) feeding and intermittent fasting are both known to prevent overweight and adiposity, improve glucose tolerance, and decrease plasma cholesterol in high-fat diet-induced obese mice. Here we examined the overall effects of TR feeding of a Western diet (fat, 40.5 Kcal%; cholesterol, 0.21 g%) using 8-week-old Apoe−/− mice. Mice were assigned into three groups: (1) an ad libitum (AL) group fed an AL Western diet, (2) a TR group with restricted access to a Western diet (15 h/day, 12:00 to 3:00 Zeitgeber time [ZT]); and (3) an Ex/TR group fed a TR Western diet and subjected to physical exercise at 12:00 ZT. Mice in the AL group gained body weight rapidly during the 14-week observation period. With TR feeding, excessive weight gain, liver adiposity, visceral fat, and brown adipose tissue volume were effectively suppressed. Although TR feeding failed to decrease Oil Red O-stained aortic plaques in Apoe−/− mice, physical exercise significantly decreased them. Neither TR feeding with exercise nor that without exercise decreased the mean area under the curve of the plasma cholesterol level or the fasting plasma glucose. Collectively, TR feeding of a Western diet prevented the development of obesity but failed to ameliorate atherosclerosis in Apoe−/− mice.

Estrogen-SIRT1 Axis Plays a Pivotal Role in Protecting Arteries Against Menopause-Induced Senescence and Atherosclerosis.

Aim: Menopause causes arterial senescence and atherosclerotic development through decrease of estrogen. Recently, histone deacetylase SIRT1 has been reported to have protective effects against arterial senescence and atherosclerosis. However, the relationship between estrogen and SIRT1 in the context of menopause-induced arterial senescence is not well understood. The present study aims to investigate whether SIRT1 is involved in the etiology of menopause-induced arterial senescence and atherosclerotic development.Methods: Twelve-week old female apolipoprotein E-knockout (ApoE-KO) mice underwent ovariectomy (OVX) or sham surgery.Results: SIRT1 expression and endothelial nitric oxide synthase (eNOS) activation in the aorta were significantly lower in OVX mice than they were in sham mice (OVX vs. sham, n = 5 per group). Senescence-associated β-galactosidase activity, protein expression of Ac-p53 and PAI-1, and aortic atherosclerosis lesions were significantly greater in OVX mice than they were in sham mice. Administration of 17β-estradiol (E2) for eight weeks to OVX mice restored aortic SIRT1 expression, activated eNOS, and retarded OVX-induced arterial senescence and atherosclerotic development (E2 vs. control, n = 5 per group). The effects of E2 on SIRT1 upregulation, anti-senescence and anti-atherosclerosis were attenuated by administration of a SIRT1 inhibitor, sirtinol. In vitro experiment using human endothelial cells demonstrated that E2 also increased SIRT1 expression and retarded oxidized low density lipoprotein-induced premature senescence, which were also abolished by sirtinol. These results suggested that estrogen modulated arterial senescence and atherosclerosis through SIRT1. Additionally a selective estrogen receptor modulator (SERM), bazedoxifene, also augmented SIRT1 and inhibited arterial senescence and atherosclerotic development (SERM vs. control, n = 3 per group).Conclusions: Downregulation of SIRT1 causes OVX-induced arterial senescence and atherosclerosis in ApoE-KO mice. Administration of estrogen or SERM enables OVX mice to restore these alterations by SIRT1 induction.

Curcumin Protects against Atherosclerosis in Apolipoprotein E-Knockout Mice by Inhibiting Toll-like Receptor 4 Expression.

Toll-like receptor 4 (TLR4) has been reported to play a critical role in the pathogenesis of atherosclerosis, the current study aimed to investigate whether curcumin suppresses atherosclerosis development in ApoE-knockout (ApoE-/-) mice by inhibiting TLR4 expression. ApoE-/- mice were fed a high-fat diet supplemented with or without curcumin (0.1% w/w) for 16 weeks. Curcumin supplementation significantly reduced TLR4 expression and macrophage infiltration in atherosclerotic plaques. Curcumin also reduced aortic interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression, nuclear factor-κB (NF-κB) activity, and plasma IL-1β, TNF-α, soluble VCAM-1 and ICAM-1 levels. In addition, aortic sinus sections revealed that curcumin treatment reduced the extent of atherosclerotic lesions and inhibited atherosclerosis development. In vitro, curcumin inhibited NF-κB activation in macrophages and reduced TLR4 expression induced by lipopolysaccharide. Our results indicate that curcumin protects against atherosclerosis at least partially by inhibiting TLR4 expression and its related inflammatory reaction.

Dietary flaxseed oil improved western-type diet-induced atherosclerosis in apolipoprotein-E knockout mice

Flaxseed oil is a rich source of n-3 fatty acid alpha-linolenic acid which has been identified as having significant cardioprotective effects. The objective of this study was to investigate the effect of partial replacement of lard with flaxseed oil on atherosclerosis and investigate the underlying mechanisms. Apolipoprotein-E knockout mice were given a normal chow diet, a lard based western-type high-fat diet (WTD), or a WTD with partial replacement of lard with 10% flaxseed oil (w/w) for 16 weeks, respectively. Results demonstrated that partial replacing of lard with flaxseed oil significantly ameliorated atherosclerosis, as well as improved lipid abnormalities, oxidative stress, and inflammation. These data were associated with modification effects on expression of genes involved in lipid metabolism (SREBP-2, HMGCR, SREBP-1c, and ACC), oxidative stress (NADPH oxidase), and inflammation (TNF-α, IL-6, MCP-1, and VCAM-1). Our data providing evidence that flaxseed oil could be a promising functional food in cardiovascular health promotion.

Pravastatin attenuates the action of the ETS domain-containing protein ELK1 to prevent atherosclerosis in apolipoprotein E-knockout mice via modulation of extracellular signal-regulated kinase 1/2 signal pathway.

Oxidative stress plays an important role in atherosclerosis, a vascular disease with high morbidity and mortality. The ETS domain-containing protein ELK1 is an oxidative stress-sensitive factor modulated by the extracellular signal-regulated kinase (ERK) 1/2 pathway. However, the role of ELK1 in the prevention of atherosclerosis by pravastatin remains unclear. In the present study, male apolipoprotein E-knockout (apoE-/- ) mice fed a diet containing 1.25% cholesterol (w/w) were divided into two groups, one treated with pravastatin (80mg/kg, 2-2.4mg/mouse per day) for 8weeks and the other not. Male C57BL/6J mice fed with a normal diet were used as a control group. Human umbilical vein endothelial cells (HUVEC) were cultured and treated with pravastatin (10μmol/L) for 18hours before testing for the presence or absence of 100μmol/L H2 O2 (24hours). Examination of pathological sections from mice aortas revealed that pravastatin treatment almost prevented atherosclerotic plaque formation. Pravastatin also inhibited increases in serum and aortic levels of oxidized low-density lipoprotein and aortic malondialdehyde levels and decreases in aortic reduced glutathione, and the activities of superoxide dismutase, catalase and glutathione peroxidase. H2 O2 -induced increases in reactive oxygen species in HUVECs were reversed by pravastatin by 48%. Pravastatin blocked the phosphorylation of ELK1 and ERK1/2 proteins and reduced mRNA levels of early growth response 1, a known atherogenic transcription factor upregulated by the ROS/ERK/ELK1 pathway, in mice. In conclusion, pravastatin attenuates the action of ELK1 induced by oxidative stress to prevent atherosclerosis, which is dependent partly on modulation of ERK1/2 signalling.

Increased expression of chitinase 3-like 1 in aorta of patients with atherosclerosis and suppression of atherosclerosis in apolipoprotein E-knockout mice by chitinase 3-like 1 gene silencing.

Introduction. The purpose of this study was to investigate the changes of chitinase 3-like 1 (CHI3L1) in the aorta of patients with coronary atherosclerosis and to determine whether inhibition of CHI3L1 by lentivirus-mediated RNA interference could stabilize atherosclerotic plaques in apolipoprotein E-knockout (ApoE−/−) mice. Methods. We collected discarded aortic specimens from patients undergoing coronary artery bypass graft surgery and renal arterial tissues from kidney donors. A lentivirus carrying small interfering RNA targeting the expression of CHI3L1 was constructed. Fifty ApoE−/− mice were divided into control group and CHI3L1 gene silenced group. A constrictive collar was placed around carotid artery to induce plaques formation. Then lentivirus was transfected into carotid plaques. Results. We found that CHI3L1 was overexpressed in aorta of patients with atherosclerosis and its expression was correlated with the atherosclerotic risk factors. After lentivirus transduction, mRNA and protein expression of CHI3L1 were attenuated in carotid plaques, leading to reduced plaque content of lipids and macrophages, and increased plaque content of collagen and smooth muscle cells. Moreover, CHI3L1 gene silencing downregulated the expression of local proinflammatory mediators. Conclusions. CHI3L1 is overexpressed in aorta from patients with atherosclerosis and the lentivirus-mediated CHI3L1 gene silencing could represent a new strategy to inhibit plaques progression.

Triacetyl-3-hydroxyphenyladenosine, a derivative of cordycepin, attenuates atherosclerosis in apolipoprotein E-knockout mice

The cholesterol-modulating, immune-regulating and anti-inflammatory properties of cordycepin are well documented. Here we examined the effects of triacetyl-3-hydroxyphenyladenosine (THPA), a derivative of cordycepin, on the development of atherosclerosis (AS) in apolipoprotein E-knockout (apoE(-/-)) mice. The atherosclerotic lesion formation displayed by the oil red O staining-positive area was reduced significantly in either the aortic root section or the whole aorta en face in THPA-administrated apoE(-/-) mice. Plasma analysis by enzymatic method or enzyme-linked immunosorbent assay (ELISA) showed that high-density lipoprotein-cholesterol (HDL-C) was decreased, whereas apolipoprotein A-I (apoA-I) levels were markedly increased by THPA. In addition, ELISA and spectrophotometric measurement showed that plasma levels of tumor necrosis factor-α, interleukin-1 and malondialdehyde were decreased in mice treated with THPA. Realtime polymerase chain reaction detection disclosed that the expression of several transporters involved in reverse cholesterol transport was induced by THPA, and the expression of hepatic ABCA1 and apoA-I, which play roles in the maturation of HDL-C, was also elevated in the THPA-treated groups. Moreover, THPA enhanced the expression of endothelial nitric oxide synthase (NOS), and reduced the expression of inducible NOS and lectin-like oxidized LDL receptor-1 in the aorta, suggesting that THPA can exert endothelial protection effects. In addition, the expression or activation of several proinflammatory factors in the aorta was suppressed by THPA. In conclusion, our results reveal the inhibitory effects of THPA on AS in apoE(-/-) mice.

Ultrasound biomicroscopy assessment of atherosclerosis in apolipoprotein-E knockout mice

ObjectivesThe purpose of this study was to validate the hypothesis high-resolution ultrasound biomicroscopy (UBM) can observe that atherosclerotic (AS) progression of invivo mice can be suppressed by IL-1Ra.MethodsTwo different age...

Noninvasive Assessment of Atherosclerosis in Apolipoprotein-E Knockout Mice by Ultrasound Biomicroscopy

Intima media thickness is a marker for human atherosclerosis. This study aimed to validate the hypothesis that atherosclerosis progression in vivo in mice can be visualized noninvasively using high-resolution ultrasound biomicroscopy (UBM), and to study the association between UBM characteristics and plasma lipids in the apolipoprotein-E knockout (ApoE-/-) mouse model. Four age groups of male ApoE-/- mice were used as atherosclerotic models, with age-matched male C57BL/6 mice used as controls. Plaque thickness and area measured by UBM correlated with histologic measurements (r = 0.81, r = 0.70, respectively; p < 0.001). Serum total cholesterol, low density lipoprotein cholesterol and triglycerides were higher in the ApoE-/- groups compared with controls ( p < 0.01). Plaque thickness was correlated with total cholesterol (r = 0.505, p < 0.001). High-resolution UBM provides a noninvasive, accurate means of detecting atherosclerosis progression in vivo in mice and can detect changes in the early stage of atherosclerosis.

Effect of nano-TiO(2) intratracheal instillation on lipid metabolism of AopE gene-knockout mice

To investigate the effect of nano-TiO(2) intratracheal instillation on the progression of dyslipidemia and atherosclerosis in apolipoprotein E-knockout mice. The nano-TiO(2) was ultrasound with phosphate-buffered saline solutions (PBS) into its suspension for exposure. A total of 46 specific pathogen free (SPF) level of 11-week-old male apolipoprotein E-knockout mice were randomly divided into groups by their body weights: non-treatment group (8 mice), PBS control group (9 mice), high dose group (1.0 mg/ml, 10 mice), medium dose group (0.5 mg/ml, 10 mice), and low dose group (0.1 mg/ml, 9 mice). Except the non-treatment group, mice from other groups were intratracheally instilled with 0.05 ml each time, twice a week. After exposure of 6 weeks, viscera index, blood TC, TG, HDL-C, LDL-C, and organic lipid ratio were assessed as biomarkers. Artery and aortic root issues were assessed by histopathology. After 5 weeks exposure, mice body weights in high dose group ((29.7 ± 1.9) g) started to drop, compared to PBS control ((31.3 ± 1.9) g, t = -1.58, P < 0.05) and low dose group ((31.4 ± 1.4) g, t = -1.17, P < 0.05); after 6 weeks, high dose group ((28.8 ± 1.5) g) was lower than PBS control ((30.4 ± 1.9) g, t = -1.60, P < 0.05), non-treatment group ((30.2 ± 1.3) g, t = -1.43, P < 0.05) and low dose group ((30.6 ± 1.0) g, t = -1.83, P < 0.05). TC levels of non-treatment, PBS control, high dose group, medium dose group and low dose group were (2.92 ± 1.18), (3.12 ± 0.73), (4.19 ± 1.86), (3.46 ± 0.72) and (2.57 ± 0.64) mmol/L, respectively; TG levels were (0.39 ± 0.13), (0.39 ± 0.08), (0.60 ± 0.21), (0.55 ± 0.19) and (0.41 ± 0.11) mmol/L, respectively; HDL-C levels were (1.67 ± 0.45), (1.54 ± 0.67), (0.93 ± 0.50), (1.02 ± 0.48) and (1.31 ± 0.64) mmol/L; TG levels of high dose group were higher than that of non-treatment group (t = 1.27, P = 0.03) and low dose group (t = 1.62, P = 0.01); TG levels of medium dose group was higher than PBS control (t = 0.16, P = 0.04), and TC levels of high dose group were higher than PBS control (t = 0.22, P = 0.01), non-treatment group (t = 0.22, P = 0.04) and low dose group (t = 0.20, P = 0.03), and HDL-C levels of high dose group were lower than PBS control (t = -0.61, P = 0.04) and non-treatment group (t = -0.74, P = 0.04); organic lipid ratio of each group were (2.27 ± 0.51)%, (2.06 ± 0.53)%, (2.90 ± 0.50)%, (2.60 ± 0.23)%, (2.24 ± 0.45)%; high dose group were higher than PBS control (t = 0.85, P = 0.00), non-treatment group (t = 0.64, P = 0.03) and low dose group (t = 0.67, P = 0.01); medium dose group was higher than PBS control (t = 0.54, P = 0.02). The plaque lipid content and calcium content which showed the progression of atherosclerosis and plaque rupture were elevated in medium and high dose groups. Intratracheal instillation of nano-TiO(2) can induce dyslipidemia and accelerate the development of atherosclerosis and plaque rupture in ApoE-/-mice.

Total panax notoginsenosides prevent atherosclerosis in apolipoprotein E-knockout mice: Role of downregulation of CD40 and MMP-9 expression

Total panax notoginsenosides (TPNS) are the main active ingredients in San-Chi, the root of Panax notoginseng (Burk) F.H. Chen, which belongs to the Araliaceae family and has been used in traditional Chinese medicine to treat atherosclerosis. We investigated the effect of TPNS on serum lipid levels and cell differentiation antigen 40 (CD40) and matrix metalloproteinase 9 (MMP-9) expression in atherosclerosis in apolipoprotein E-knockout (apoE-KO) mice fed a high-fat, high-cholesterol diet. Twenty-four apoE-KO mice were divided into two groups, the ApoE-KO group and the ApoE-KO + TPNS group. TPNS (60 mg/kg) was orally administered daily for 12 weeks in ApoE-KO + TPNS group. After 12 weeks, blood and aortas were obtained. Serum levels of lipid were analyzed, serum oxidized low density lipoprotein (oxLDL) concentration, ratio of plaque area-to-vessel area and the expression of CD40 and MMP-9 were examined by ELISA, histological staining, immunohistochemistry and real-time PCR, respectively. It was observed in our study that serum levels of lipid and oxLDL, ratio of plaque area to vessel area, and expression of CD40 and MMP-9 were lower in the ApoE-KO + TPNS group than in the ApoE-KO group. These results suggest that TPNS could prevent atherosclerosis by lowering serum lipid levels and regulating vascular CD40 and MMP-9 expression. TPNS may have implications for clinical treatment of atherosclerosis vascular disease.

Arsenic and Cardiovascular Disease

Chronic arsenic exposure is a worldwide health problem. Although arsenic-induced cancer has been widely studied, comparatively little attention has been paid to arsenic-induced vascular disease. Epidemiological studies have shown that chronic arsenic exposure is associated with increased morbidity and mortality from cardiovascular disease. In addition, studies suggest that susceptibility to arsenic-induced vascular disease may be modified by nutritional factors in addition to genetic factors. Recently, animal models for arsenic-induced atherosclerosis and liver sinusoidal endothelial cell dysfunction have been developed. Initial studies in these models show that arsenic exposure accelerates and exacerbates atherosclerosis in apolipoprotein E-knockout mice. Microarray studies of liver mRNA and micro-RNA abundance in mice exposed in utero suggest that a permanent state of stress is induced by the arsenic exposure. Furthermore, the livers of the arsenic-exposed mice have activated pathways involved in immune responses suggesting a pro-hyperinflammatory state. Arsenic exposure of mice after weaning shows a clear dose-response in the extent of disease exacerbation. In addition, increased inflammation in arterial wall is evident. In response to arsenic-stimulated oxidative signaling, liver sinusoidal endothelium differentiates into a continuous endothelium that limits nutrient exchange and waste elimination. Data suggest that nicotinamide adenine dinucleotide phosphate oxidase-derived superoxide or its derivatives are essential second messengers in the signaling pathway for arsenic-stimulated vessel remodeling. The recent findings provide future directions for research into the cardiovascular effects of arsenic exposure.

Chronic urotensin II infusion enhances macrophage foam cell formation and atherosclerosis in apolipoprotein E-knockout mice

Our recent studies have indicated that urotensin II, the most potent vasoconstrictor peptide identified to date, potentiates human macrophage foam cell formation and vascular smooth muscle cell proliferation, and its levels are increased in the plasma of hypertensive patients with carotid atherosclerotic plaques. In the present study, we investigated the enhancing effect of urotensin II on atherosclerosis in apolipoprotein E-knockout mice and its suppression by 4-aminoquinoline, an urotensin II receptor-selective antagonist. Urotensin II, urotensin II + 4-aminoquinoline, or vehicle was infused for 4 weeks through an osmotic mini-pump into 9-week-old apolipoprotein E-knockout mice on a high-fat diet. Aortic atherosclerosis and foam cell formation in exudate peritoneal macrophages were examined. Atherosclerotic lesions as well as plasma levels of urotensin II, reactive oxygen species, and oxidized low-density lipoprotein and oxidized low-density lipoprotein-induced foam cell formation were significantly greater in urotensin II-infused mice than vehicle-infused controls. Western blotting analysis showed increased expression of scavenger receptors (CD36 and scavenger receptor class A) and acyl-CoA:cholesterol acyltransferase-1 in these macrophages. Increases in these parameters were significantly reduced by addition of 4-aminoquinoline. In apolipoprotein E-knockout mice even without urotensin II infusion, the treatment with 4-aminoquinoline for 8 weeks significantly prevented the development of atherosclerotic lesions. Our results provide the first evidence that increased plasma urotensin II level stimulates oxidized low-density lipoprotein and reactive oxygen species production and macrophage foam cell formation via increased expression of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1, contributing to the development of atherosclerosis in apolipoprotein E-deficient mice. Urotensin II receptor antagonism may be a promising therapeutic strategy against atherosclerosis.

Angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis

The endothelium plays a central role in the maintenance of vascular homeostasis. One of the main effectors of endothelial dysfunction is ANG II, and pharmacological approaches to limit ANG II bioactivity remain the cornerstone of cardiovascular therapeutics. Angiotensin converting enzyme-2 (ACE2) has been identified as a critical negative modulator of ANG II bioactivity, counterbalancing the effects of ACE in determining net tissue ANG II levels; however, the role of ACE2 in the vasculature remains unknown. In the present study, we hypothesized that ACE2 is a novel target to limit endothelial dysfunction and atherosclerosis. To this aim, we performed in vitro gain and loss of function experiments in endothelial cells and evaluated in vivo angiogenesis and atherosclerosis in apolipoprotein E-knockout mice treated with AdACE2. ACE2-deficient mice exhibited impaired endothelium-dependent relaxation. Overexpression of ACE2 in human endothelial cells stimulated endothelial cell migration and tube formation, and limited monocyte and cellular adhesion molecule expression; effects that were reversed in ACE2 gene silenced and endothelial cells isolated from ACE2-deficient animals. ACE2 attenuated ANG II-induced reactive oxygen species production in part through decreasing the expression of p22phox. The effects of ACE2 on endothelial activation were attenuated by pharmacological blockade of ANG-(1-7) with A779. ACE2 promoted capillary formation and neovessel maturation in vivo and reduced atherosclerosis in apolipoprotein E-knockout mice These data indicate that ACE2, in an ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis via a mechanism that may involve attenuation of NADPHox-induced reactive oxygen species production. ACE2-based treatment approaches may be a novel approach to limit aberrant vascular responses and atherothrombosis.

877. Differential Expression of Indoleamine 2,3-dioxygenase (IDO) by Endothelial Cells and Its Overexpression by Gene Transfer: Implications for Cardiovascular Biology

Background: Indoleamine 2,3-dioxygenase (IDO) is an important enzyme in the regulation of immune responses. The cells that express IDO can suppress T cell responses and promote tolerance. We have investigated the role of IDO expression by vascular endothelial cells and its overexpression by gene transfer and its consequence on immunoregulation. Many studies have now indicated that in atherosclerosis, there is a defect in the immunoregulation1. Thus far, very few investigations have been conducted to modify this defective component of immune system. We have therefore explored the role of IDO following gene transfer on immune system with a long-term goal to modulate alloresponse or atherosclerotic process. Methods ? Results: We compared the expression of IDO by primary human umbilical vein endothelial cells (HUVECs) (fetal tissues) and human saphenous vein endothelial cells (HSVECs) (adult tissues) using reverse transcriptase PCR, Western blotting and assays for enzymatic activity. IDO is constitutively expressed by HUVECs, and can be upregulated by incubation with cytokines. On the other hand, HSVEC express little IDO, which is poorly upregulated upon activation (except by mycoplasma). Inhibition of IDO activity improved the ability of HUVEC to stimulate allogeneic T cell responses. If either HUVEC or HSVEC are transfected with the gene encoding IDO then they are incapable of stimulating allogeneic T cell responses, and induce anergy in allospecific T cells, which can also act as regulatory cells. Using our immunolipoplexes targeting specifically against vascular EC 2,3, IDO construct was successfully transfected into HUVEC and HSVEC. This resulted in a functional effect on T cell tolerance induction. These data indicate that IDO is a good therapeutic gene to modulate rejection process or to modulate that the immune components seen in the chronic atherosclerosis. Conclusion: The variable expression of IDO in different endothelial cells is important not only in understanding the role of endothelial cells in the regulation of immune responses, but also as a potential therapeutic strategy through a gene-based treatment to modulate the alloresponse or the immune component of the atherosclerosis.Reference:1. Mallat, Z. et al. Induction of a regulatory T cell type 1 response reduces the development of atherosclerosis in apolipoprotein E-knockout mice. Circulation108, 1232–7 (2003).2. Tan, P. H. et al. Antibody targeted gene transfer to endothelium. J Gene Med5, 311–323 (2003).3. Tan, P. H. et al. Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination. Mol Ther11, 790–800 (2005). Background: Indoleamine 2,3-dioxygenase (IDO) is an important enzyme in the regulation of immune responses. The cells that express IDO can suppress T cell responses and promote tolerance. We have investigated the role of IDO expression by vascular endothelial cells and its overexpression by gene transfer and its consequence on immunoregulation. Many studies have now indicated that in atherosclerosis, there is a defect in the immunoregulation1. Thus far, very few investigations have been conducted to modify this defective component of immune system. We have therefore explored the role of IDO following gene transfer on immune system with a long-term goal to modulate alloresponse or atherosclerotic process. Methods ? Results: We compared the expression of IDO by primary human umbilical vein endothelial cells (HUVECs) (fetal tissues) and human saphenous vein endothelial cells (HSVECs) (adult tissues) using reverse transcriptase PCR, Western blotting and assays for enzymatic activity. IDO is constitutively expressed by HUVECs, and can be upregulated by incubation with cytokines. On the other hand, HSVEC express little IDO, which is poorly upregulated upon activation (except by mycoplasma). Inhibition of IDO activity improved the ability of HUVEC to stimulate allogeneic T cell responses. If either HUVEC or HSVEC are transfected with the gene encoding IDO then they are incapable of stimulating allogeneic T cell responses, and induce anergy in allospecific T cells, which can also act as regulatory cells. Using our immunolipoplexes targeting specifically against vascular EC 2,3, IDO construct was successfully transfected into HUVEC and HSVEC. This resulted in a functional effect on T cell tolerance induction. These data indicate that IDO is a good therapeutic gene to modulate rejection process or to modulate that the immune components seen in the chronic atherosclerosis. Conclusion: The variable expression of IDO in different endothelial cells is important not only in understanding the role of endothelial cells in the regulation of immune responses, but also as a potential therapeutic strategy through a gene-based treatment to modulate the alloresponse or the immune component of the atherosclerosis. Reference: 1. Mallat, Z. et al. Induction of a regulatory T cell type 1 response reduces the development of atherosclerosis in apolipoprotein E-knockout mice. Circulation108, 1232–7 (2003). 2. Tan, P. H. et al. Antibody targeted gene transfer to endothelium. J Gene Med5, 311–323 (2003). 3. Tan, P. H. et al. Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination. Mol Ther11, 790–800 (2005).

Gene therapy targeting monocyte chemoattractant protein-1 for vascular disease.

Monocyte chemoattractant protein-1 (MCP-1) has been shown to play an essential role in the pathogenesis of arteriosclerosis and other vascular diseases, such as restenosis after arterial injury, by recruiting monocytes into the arterial wall. We devised a new strategy for anti-MCP-1 gene therapy against arteriosclerosis by transfecting an amino-terminal deletion mutant (7ND), which lacks the amino-terminal amino acids 2 to 8 of the human MCP-1 gene, into a remote organ (skeletal muscles). Intramuscular transduction with the mutant MCP-1 gene suppressed inflammatory and proliferative changes and arteriosclerosis formation induced by the chronic inhibition of nitric oxide synthesis in rats. 7ND gene transfection also inhibited the initiation, progression, and destabilization of atherosclerosis in Apolipoprotein E-knockout mice. Moreover, the strategy reduced restenosis after balloon injury in rabbits, rats, and monkeys, or neointimal formation after stent implantation in monkeys. This new strategy may be a useful and feasible gene therapy against atherosclerosis and restenosis after angioplasty.

Cytomegalovirus infection increases development of atherosclerosis in Apolipoprotein-E knockout mice

Cytomegalovirus (CMV) infection has been associated with coronary artery disease, but it is unknown whether the virus can causally contribute to atherogenesis. To determine whether the virus has this capacity, we infected an atherosclerotic-prone mouse strain (C57BL/6J apoE−/−) with murine CMV. At 14 days of age, 30 mice received CMV (30 000 pfu) ip and 30 received virus free media. At 13 and 16 weeks atherosclerotic lesion size was measured from aortic sinus cross-sections. Infection did not alter plasma levels of cholesterol, triglycerides, and high density lipoprotein (HDL); however, 4 weeks after infection IFNγ levels were elevated (infection vs control: 156 ± 49 vs 50 ± 22 pg/ml, P = 0.04). No differences in lesion size were present at 13 weeks post infection. However, by 16 weeks mean aortic sinus lesion area (mm 2 × 10 3 ± SEM; N = 75) in the CMV-infected mice was significantly greater than in uninfected mice (74 ± 6 vs 57 ± 6; P = 0.04). CMV caused the greatest increase (34%) in lesion size in females (103 ± 9 vs 77 ± 10; P = 0.05; N = 35). These results provide additional evidence implicating CMV as a causal agent of atherosclerosis, at least in an animal model.