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1.  AIP1 suppresses atherosclerosis by limiting hyperlipidemia-induced inflammation and vascular endothelial dysfunction 
Apoptosis signal-regulating kinase 1 (ASK1)–interacting protein-1 (AIP1) is a signaling adaptor molecule implicated in stress and apoptotic signaling induced by proinflammatory mediators. However, its function in atherosclerosis has not been established. In the present study, we use AIP1-null (AIP1−/−) mice to examine its effect on atherosclerotic lesions in an ApoE-null (ApoE−/−) mouse model of atherosclerosis.
ApoE−/− control mice developed atherosclerosis in the aortic roots and descending aortas upon Western-type diet for 10 weeks, while the atherosclerotic lesions are significantly augmented in ApoE−/−AIP1−/− double knockout (DKO) mice. DKO mice show increases in plasma inflammatory cytokines with no significant alterations in body weight, total cholesterol levels or lipoprotein profiles. Aortas in DKO mice show increased inflammation and endothelial cell (EC) dysfunction with NF-κB activity, correlating with increased accumulation of macrophages in the lesion area. Importantly, macrophages from DKO donors are not sufficient to augment inflammatory responses and atherogenesis when transferred to ApoE-KO recipients. Mechanistic studies suggest that AIP1 is highly expressed in aortic EC but not in macrophages, and AIP1 deletion in EC significantly enhance oxidized LDL-induced NF-κB signaling, gene expression of inflammatory molecules and monocyte adhesion, suggesting that vascular EC are responsible for the increased inflammatory responses observed in DKO mice.
Our data demonstrate that loss of AIP1 in aortic EC primarily contributes to the exacerbated lesion expansion in the ApoE−/−AIP1−/− mice, revealing an important role of AIP1 in limiting inflammation, EC dysfunction and atherosclerosis.
PMCID: PMC3637885  PMID: 23413429
Atherosclerosis; inflammation; endothelial cell; lipids and lipoproteins; macrophage; AIP1
2.  Reduced Alpha-Lipoic Acid Synthase Gene Expression Exacerbates Atherosclerosis in Diabetic Apolipoprotein E–Deficient Mice 
Atherosclerosis  2012;223(1):137-143.
To study the effects of reduced lipoic acid gene expression on diabetic atherosclerosis in apolipoprotein E null mice (Apoe−/−).
Methods and Results
Heterozygous lipoic acid synthase gene knockout mice (Lias+/−) crossed with Apoe−/− mice were used to evaluate the diabetic effect induced by streptozotocin on atherosclerosis in the aortic sinus of the heart. While diabetes markedly increased atherosclerotic plaque size in Apoe−/− mice, a small but significant effect of reduced expression of lipoic acid gene was observed in diabetic Lias+/−Apoe−/− mice. In the aortic lesion area, the Lias+/−Apoe−/− mice exhibited significantly increased macrophage accumulation and cellular apoptosis than diabetic Lias+/+Apoe−/− littermates. Plasma glucose, cholesterol, and interleukin-6 were also higher. These abnormalities were accompanied with increased oxidative stress including a decreased ratio of reduced glutathione/oxidized glutathione in erythrocytes, increased systemic lipid peroxidation, and increased Gpx1 and MCP1 gene expression in the aorta.
Decreased endogenous lipoic acid gene expression plays a role in development of diabetic atherosclerosis. These findings extend our understanding of the role of antioxidant in diabetic atherosclerosis.
PMCID: PMC3389304  PMID: 22658261
lipoic acid; Lias mouse model; atherosclerosis; diabetes; and apolipoprotein E null mice
3.  Deletion of Tenascin-C gene Exacerbates Atherosclerosis and Induces Intraplaque Hemorrhage in Apo E Deficient Mice 
Tenascin-C (TNC), a matricellular protein, is upregulated in atherosclerotic plaques. We investigated whether the deletion of TNC gene affects the development of atherosclerosis in a murine model.
TNC−/−/apo E−/− mice were generated and used for atherosclerosis studies. We compared these results to those observed in control groups of apo E−/− mice.
The en face analysis of aortic area showed that the mean aortic lesion area of the double KO mice was significantly higher than control mice at different times after feeding of atherogenic diet; the accumulation of lesional macrophages and lipids were significantly higher, respectively. Analysis of cell adhesion molecules revealed that VCAM-1, but not ICAM-1, was upregulated 1 week after feeding of atherogenic diet in the double KO mouse as compared to apo E−/− mouse. Cell culture studies revealed that the expression of VCAM-1 in endothelial cells isolated from the double KO mouse is more sensitive to the TNFα stimulation than the cells isolated from apo E−/− mice. Cell adhesion studies showed that the adherence of RAW monocytic cells to the endothelial cells was significantly enhanced in the cultured endothelial cells from the TNC gene-deleted cells. Following the prolonged feeding of an atherogenic diet (28–30 weeks), the aortic and carotid atherosclerotic lesions frequently demonstrated large grossly visible areas of intraplaque hemorrhage in the double KO mice compared to control.
These data unveil a protective role for TNC in atherosclerosis and suggest that TNC signaling may have the potential to reduce atherosclerosis, in part by modulating VCAM-1 expression.
PMCID: PMC3345312  PMID: 22300502
tenascin; atherosclerosis; plaque hemorrhage; extracellular matrix; VCAM-1
4.  Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of Atherosclerosis in Diabetes 
Diabetes  2008;57(9):2461-2469.
OBJECTIVE—Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE−/− model of accelerated atherosclerosis.
RESEARCH DESIGN AND METHODS—ApoE−/− and RAGE−/−/apoE−/− double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis.
RESULTS—Although diabetic apoE−/− mice showed increased plaque accumulation (14.9 ± 1.7%), diabetic RAGE−/−/apoE−/− mice had significantly reduced atherosclerotic plaque area (4.9 ± 0.4%) to levels not significantly different from control apoE−/− mice (4.3 ± 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-κB subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE−/− mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE−/−/apoE−/− mice.
CONCLUSIONS—This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications.
PMCID: PMC2518498  PMID: 18511846
5.  Omega-3 Fatty Acids Ameliorate Atherosclerosis by Favorably Altering Monocyte Subsets and Limiting Monocyte Recruitment to Aortic Lesions 
Fish oil (FO), containing n-3 fatty acids (FAs), attenuates atherosclerosis. We hypothesized that n-3 FA-enriched oils are atheroprotective through alteration of monocyte subsets and their trafficking into atherosclerotic lesions.
Methods and Results
Low density lipoprotein receptor knockout (LDLr−/−) and apolipoprotein E−/− (apoE) mice were fed diets containing 10% (calories) as palm oil (PO) and 0.2% cholesterol, supplemented with an additional 10% PO, echium oil (EO; containing 18:4 n-3) or FO. Compared to PO-fed LDLr−/− mice, EO and FO significantly reduced plasma cholesterol, splenic Ly6Chi monocytosis by ~50%, atherosclerosis by 40–70%, monocyte trafficking into the aortic root by ~50%, and atherosclerotic lesion macrophage content by 30–44%. In contrast, atherosclerosis and monocyte trafficking into the artery wall was not altered by n-3 FAs in apoE−/− mice; however, Ly6Chi splenic monocytes positively correlated with aortic root intimal area across all diet groups. In apoE−/− mice, FO reduced the percentage of blood Ly6Chi monocytes, despite an average two-fold higher plasma cholesterol relative to PO.
The presence of splenic Ly6Chi monocytes parallels the appearance of atherosclerotic disease in both LDLr−/− and apoE−/− mice. Furthermore, n-3 FAs favorably alter monocyte subsets independently from effects on plasma cholesterol, and reduce monocyte recruitment into atherosclerotic lesions.
PMCID: PMC3431287  PMID: 22814747
monocytosis; n-3 fatty acids; fish oil; Echium oil; inflammation; Ly6C
6.  Genetic deletion of chemokine receptor Ccr7 exacerbates atherogenesis in ApoE-deficient mice 
Cardiovascular Research  2012;97(3):580-588.
Recent evidence suggests that both Ccr7 and its ligands, Ccl19 and Ccl21, are present in mouse and human atherosclerotic plaques; however, the role of Ccr7 in atherogenesis is still controversial. Here, we addressed this question by using the classic apolipoprotein E-deficient (ApoE−/−) mouse model of atherosclerosis.
Methods and results
Ccr7−/−ApoE−/− double knockout mice and Ccr7+/+ApoE−/− littermates were generated and maintained on a high-fat Western diet for 8 weeks to induce atherosclerosis. Ccr7−/−ApoE−/− mice showed an ∼80% increase in atherosclerotic lesion size in the whole aorta and a two-fold increase in the aortic root compared with Ccr7+/+ApoE−/− mice. Ccr7−/−ApoE−/− mice had increased T cells in the blood, bone marrow, and spleen, as well as in atherosclerotic lesions. Competitive repopulation experiments revealed that T cells from Ccr7−/−ApoE−/− mice migrated poorly into lymph nodes but better into mouse aortas compared with T cells from Ccr7+/+ApoE−/− mice. Transplantation of the bone marrow from Ccr7−/−ApoE−/− mice into lethally irradiated Ccr7+/+ApoE−/− mice resulted in ∼60% more atherosclerotic lesions compared with Ccr7+/+ApoE−/− donor bone marrow, suggesting that exacerbation was mediated by a Ccr7+ bone marrow-derived cell(s). Furthermore, in Ccr7−/−ApoE−/− mice the serum level of IL-12 was markedly increased, whereas the level of transforming growth factor beta (TGF-β) was significantly decreased, suggesting an imbalance of T cell responses in these mice.
Our data suggest that genetic deletion of Ccr7 exacerbates atherosclerosis by increasing T cell accumulation in atherosclerotic lesions.
PMCID: PMC3567784  PMID: 23180724
Atherosclerosis; Inflammation; Leucocytes; Aorta
7.  Cardiac and vascular changes in elderly atherosclerotic mice: the influence of gender 
Although advanced age is considered a risk factor for several diseases, the impact of gender on age-associated cardiovascular diseases, such as atherosclerotic processes and valvular diseases, remains not completely clarified. The present study was designed to assess aortic valve morphology and function and vascular damage in elderly using the apolipoprotein E knockout (ApoE KO) mouse. Our hypothesis was that advanced age-related cardiovascular changes are aggravated in atherosclerotic male mice.
The grade (0 to 4) of aortic regurgitation was evaluated through angiography. In addition, vascular lipid deposition and senescence were evaluated through histochemical analyses in aged male and female ApoE KO mice, and the results were compared to wild-type C57BL/6J (C57) mice.
Aortic regurgitation was observed in 92% of the male ApoE KO mice and 100% of the male C57 mice. Comparatively, in age-matched female ApoE KO and C57 mice, aortic regurgitation was observed in a proportion of 58% and 53%, respectively. Histological analysis of the aorta showed an outward (positive) remodeling in ApoE KO mice (female: 1.86 ± 0.15; male: 1.89 ± 0.68) using C57 groups as reference values. Histochemical evaluation of the aorta showed lipid deposition and vascular senescence only in the ApoE KO group, which were more pronounced in male mice.
The data show that male gender contributes to the progression of aortic regurgitation and that hypercholesterolemia and male gender additively contribute to the occurrence of lipid deposition and vascular senescence in elderly mice.
PMCID: PMC2936359  PMID: 20723257
8.  Mitogen-Activated Protein Kinase Phosphatase-1 Deficiency Decreases Atherosclerosis in Apolipoprotein E Null Mice by Reducing Monocyte Chemoattractant Protein-1 Levels 
We previously reported that mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is necessary for oxidized phospholipids to induce monocyte chemoattractant protein-1 (MCP-1) secretion by human aortic endothelial cells. We also reported that inhibition of tyrosine phosphatases including MKP-1 ameliorated atherosclerotic lesions in mouse models of atherosclerosis.
This study was conducted to further investigate the specific role of MKP-1 in atherogenesis.
Methods and Results
We generated MKP-1−/−/apoE−/− double-knockout mice. At 24 weeks of age, the size, macrophage and dendritic cell content of atherosclerotic lesions of the aortic root were significantly lower (~-41% for lesions and macropahges, and ~-78% for dendritic cells) in MKP-1−/−/apoE−/− mice when compared with apoE−/− mice. Total cholesterol (−18.4%, p=0.045) and very low-density lipoprotein (VLDL)/ low-density lipoprotein (LDL) (-20.0%, p=0.052) cholesterol levels were decreased in MKP-1−/−/apoE−/− mice. Serum from MKP-1−/−/apoE−/− mice contained significantly lower levels of MCP-1 and possessed significantly reduced capability to induce monocyte migration in vitro. Moreover, peritoneal macrophages isolated from MKP-1−/−/apoE−/− mice produced significantly lower levels of MCP-1 when compared to peritoneal macrophages from apoE−/− mice. Furthermore, MKP-1−/−/apoE−/− mice had significantly reduced serum hydroxyeicosatetraenoic acids (HETEs) levels, which have been reported to induce MCP-1 levels.
Our results demonstrate that MKP-1 deficiency significantly decreases atherosclerotic lesion development in mice, in part, by affecting MCP-1 levels in the circulation and MCP-1 production by macrophages. MKP-1 may serve as a potential therapeutic target for the treatment of atherosclerotic disease.
PMCID: PMC3037189  PMID: 20619710
mitogen-activated protein kinase phosphatase-1; atherosclerosis; monocyte chemoattractant protein-1; monocytes
9.  Gain and Loss of Function for Glutathione Synthesis: Impact on Advanced Atherosclerosis in Apo E Deficient Mice 
Glutamate-cysteine ligase (GCL) is the rate-limiting step in glutathione synthesis. The enzyme is a hetero-dimer composed of a catalytic subunit GCLC and a modifier subunit GCLM.
We generated apo E−/− mice deficient in GCLM (apoE−/−/Gclm−/−) and transgenic mice that over-express GCLC specifically in macrophages (apoE−/−/Gclc-Tg) to test the hypothesis that significantly altering the availability of glutathione has a measurable impact on both the initiation and progression of atherosclerosis.
Methods and Results
Atherosclerotic plaque size and composition were measured in the innominate artery in chow-fed male and female mice at 20, 30, 40 and 50 weeks of age and in the aortic sinus at 40 or 50 weeks of age. The apoE−/−/Gclm−/− mice more rapidly developed complex lesions while the apoE−/−/Gclc-Tg mice had reduced lesion development as compared to the littermate apo E−/− control mice. Transplant of bone marrow from the apoE−/−/Gclm−/− and apoE−/−/Gclc-Tg mice into apo E−/− mice with established lesions also stimulated or inhibited further lesion development at 30 weeks post-transplant.
Gain and loss of function in the capacity to synthesize glutathione especially in macrophages has reciprocal effects on the initiation and progression of atherosclerosis at multiple sites in apo E−/− mice.
PMCID: PMC3415243  PMID: 21868708
Atherosclerosis; macrophages; glutathione; apo E−/−
10.  A Critical Role of Platelet Adhesion in the Initiation of Atherosclerotic Lesion Formation 
The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE−/− mice before the development of manifest atherosclerotic lesions. Platelet–endothelial cell interaction involved both platelet glycoprotein (GP)Ibα and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE−/− mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process.
PMCID: PMC2194025  PMID: 12370251
platelets; endothelium; atherosclerosis; integrin; glycoprotein Ib
11.  Darbepoetin Alpha Reduces Oxidative Stress and Chronic Inflammation in Atherosclerotic Lesions of Apo E Deficient Mice in Experimental Renal Failure 
PLoS ONE  2014;9(2):e88601.
Cardiovascular morbidity and mortality is very important in patients with chronic renal failure. This occurs even in mild impairment of renal function and may be related to oxidative stress and chronic inflammation. The nephrectomized apo E knockout mouse is an accepted model for evaluating atherosclerosis in renal dysfunction. Erythropoietin derivates showed anti-oxidative and anti-inflammatory effects. Therefore, this study evaluates the effects of Darbepoetin on markers of oxidative stress and chronic inflammation in atherosclerotic lesions in apo E knockout mice with renal dysfunction.
Apo E knockout mice underwent unilateral (Unx, n = 20) or subtotal (Snx, n = 26) nephrectomy or sham operation (Sham, n = 16). Mice of each group were either treated with Darbepoetin or saline solution, a part of Snx mice received a tenfold higher dose of Darbepoetin. The aortic plaques were measured and morphologically characterized. Additional immunhistochemical analyses were performed on tissue samples taken from the heart and the aorta.
Both Unx and Snx mice showed increased expression of markers of oxidative stress and chronic inflammation. While aortic plaque size was not different, Snx mice showed advanced plaque stages when compared to Unx mice. Darbepoetin treatment elevated hematocrit and lowered Nitrotyrosin as one marker of oxidative stress, inflammation in heart and aorta, plaque stage and in the high dose even plaque cholesterol content. In contrast, there was no influence of Darbepoetin on aortic plaque size; high dose Darbepoetin treatment resulted in elevated renal serum parameters.
Darbepoetin showed some protective cardiovascular effects irrespective of renal function, i.e. it improved plaque structure and reduced some signs of oxidative stress and chronic inflammation without affecting plaque size. Nevertheless, the dose dependent adverse effects must be considered as high Darbepoetin treatment elevated serum urea. Elevation of hematocrit might be a favorable effect in anemic Snx animals but a thrombogenic risk in Sham animals.
PMCID: PMC3938414  PMID: 24586350
12.  NFAT5 expression in bone marrow-derived cells enhances atherosclerosis and drives macrophage migration 
Objective: We have previously shown that the transcription factor, nuclear factor of activated T-cells 5 (NFAT5), regulates vascular smooth muscle cell phenotypic modulation, but the role of NFAT5 in atherosclerosis is unknown. Our main objective was to determine if NFAT5 expression in bone marrow (BM)-derived cells altered atherosclerotic development and macrophage function. Methods and Results: NFAT5+/−ApoE−/− mice were generated for in vivo atherosclerosis studies. Following high fat diet feeding, en face analysis of the thoracic aorta established that genome-wide NFAT5 haploinsufficiency reduced atherosclerotic lesion formation by 73%. BM transplant studies revealed that transplantation of NFAT5+/−ApoE−/− marrow into NFAT5+/+ApoE−/− mice resulted in a similar 86% reduction in lesion formation. In vitro functional analysis of BM-derived macrophages demonstrated that NFAT5 is required for macrophage migration, which is a key event in the propagation of atherosclerosis. Conclusion: We have identified NFAT5 in BM-derived cells as a positive regulator of atherosclerotic lesion formation and macrophage function in the vasculature.
PMCID: PMC3429083  PMID: 22934063
TonEBP; macrophage; atherosclerosis; bone marrow; migration
13.  Gene expression in atherosclerotic lesion of ApoE deficient mice. 
Molecular Medicine  2001;7(6):383-392.
BACKGROUND: Atherosclerosis, the major cause of mortality and invalidity in industrialized countries, is a multifactorial disease associated with high plasma cholesterol levels and inflammation in the vessel wall. Many different genes have previously been demonstrated in atherosclerosis, although limited numbers of genes are dealt with in each study. In general, data on dynamic gene expression during disease progress is limited and large-scale evaluation of gene expression patterns during atherogenesis could lead to a better understanding of the key events in the pathogenesis of atherosclerosis. We have therefore applied a mouse gene filter array to analyze gene expression in atherosclerotic ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed atherogenic western diet for 10 or 20 weeks and aortas isolated. C57BL/6 mice on normal chow were used as controls. The mRNAs of 15 animals were pooled and hybridized onto commercially available Clontech mouse gene array filters. RESULTS: The overall gene expression in the ApoE-deficient and control mice correlated well at both time points. Gene expression profiling showed varying patterns including genes up-regulated at 10 or 20 weeks only. At 20 weeks of diet, an increasing number of up-regulated genes were found in ApoE-deficient mice. CONCLUSIONS: The gene expression in atherogenesis is not a linear process with a maximal expression at advanced lesion stage. Instead, several genes demonstrate a dynamic expression pattern with peaks at the intermediate lesions stage. Thus, detailed evaluation of gene expression at several time points should help understanding the development of atherosclerosis and establishment of preventive intervention.
PMCID: PMC1950046  PMID: 11474131
14.  PSGL-1 is highly expressed on Ly-6Chi monocytes and a major determinant for Ly-6Chi monocyte recruitment to sites of atherosclerosis in mice 
Circulation  2008;117(25):3227-3237.
Ly-6Chi monocytes are key contributors to atherosclerosis in mice. However, how Ly-6Chi monocytes selectively accumulate in atherosclerotic lesions is largely unknown. Monocyte homing to sites of atherosclerosis is primarily initiated by rolling on P- and E-selectin expressed on endothelium. We hypothesize that P-selectin glycoprotein ligand-1 (PSGL-1), the common ligand of P- and E-selectin on leukocytes, contributes to the preferential homing of Ly-6Chi monocytes to atherosclerotic lesions.
Methods and Results
To test this hypothesis, we examined the expression and function of PSGL-1 on Ly-6Chi and Ly-6Clo monocytes from wild-type mice, ApoE-/- mice, and mice lacking both ApoE and PSGL-1 genes (ApoE-/-/PSGL-1-/-). We found that Ly-6Chi monocytes expressed a higher level of PSGL-1, and had enhanced binding to fluid-phase P- and E-selectin, compared to Ly-6Clo monocytes. Under in vitro flow conditions, more Ly-6Chi monocytes rolled on P-, E-, and L-selectin at slower velocities than Ly-6Clo cells. In an ex vivo perfused carotid artery model, Ly-6Chi monocytes interacted preferentially with atherosclerotic endothelium compared with Ly-6Clo monocytes in a PSGL-1-dependent manner. In vivo, ApoE-/- mice lacking PSGL-1 had impaired Ly-6Chi monocyte recruitment to atherosclerotic lesions. Moreover, ApoE-/-/PSGL-1-/- mice exhibited significantly reduced monocyte infiltration in wire injury-induced neointima and in atherosclerotic lesions. ApoE-/-/PSGL-1-/- mice also developed smaller neointima and atherosclerotic plaques.
These data indicate that PSGL-1 is a new marker for Ly-6Chi monocytes and a major determinant for Ly-6Chi cell recruitment to sites of atherosclerosis in mice.
PMCID: PMC2596619  PMID: 18519846
atherosclerosis; leukocytes; endothelium; cell adhesion molecules
15.  Pravastatin Prevents Aortic Atherosclerosis via Modulation of Signal Transduction and Activation of Transcription 3 (STAT3) to Attenuate Interleukin-6 (IL-6) Action in ApoE Knockout Mice 
The purpose of this study was to determine whether pravastatin’s prevention of aortic atherosclerosis via attenuation of IL-6 action depends on modulation of STAT3 activity. Male apoE knockout (apoE-/-) mice fed on a diet containing 1.25% cholesterol (wt/wt) were divided into pravastatin group provided with pravastatin (80 mg kg-1 per day) and atherosclerosis group. After eight weeks, pravastatin significantly prevented atherosclerotic lesion and reduced levels of IL-6 in serum and lesion, and significantly decreased expressions of phosphorylated STAT3 (pSTAT3) and increased suppressor of cytokine signaling 3 (SOCS3) expressions in lesions. Our results suggested that pravastatin’s aortic atherosclerosis preventing action via attenuation of IL-6 action may partially depend on modulation of STAT3 activity.
PMCID: PMC2635628  PMID: 19330073
Pravastatin; atherosclerosis; IL-6; STAT3; SOCS3
16.  Protective immunity against atherosclerosis carried by B cells of hypercholesterolemic mice 
Atherosclerosis is characterized by vascular inflammation and associated with systemic and local immune responses to oxidized LDL (oxLDL) and other antigens. Since immunization with oxLDL reduces atherosclerosis, we hypothesized that the disease might be associated with development of protective immunity. Here we show that spleen-associated immune activity protects against atherosclerosis. Splenectomy dramatically aggravated atherosclerosis in hypercholesterolemic apoE knockout (apoE°) mice. Transfer of spleen cells from atherosclerotic apoE° mice significantly reduced disease development in young apoE° mice. To identify the protective subset, donor spleen cells were divided into B and T cells by immunomagnetic separation before transfer. Protection was conferred by B cells, which reduced disease in splenectomized apoE° mice to one-fourth of that in splenectomized apoE° controls. Protection could also be demonstrated in intact, nonsplenectomized mice and was associated with an increase in antibody titers to oxLDL. Fewer CD4+ T cells were found in lesions of protected mice, suggesting a role for T-B cell cooperation. These results demonstrate that B cell–associated protective immunity develops during atherosclerosis and reduces disease progression.
PMCID: PMC150903  PMID: 11901183
17.  Spatial and Temporal Expression, and Statin Responsiveness of Galectin-1 and Galectin-3 in Murine Atherosclerosis 
Korean Circulation Journal  2013;43(4):223-230.
Background and Objectives
Existing data on the spatiotemporal expression patterns of a variety of galectins in murine atherosclerosis are limited. We investigated the expression levels of galectins, and their in vivo spatiotemporal expression patterns and statin responsiveness in the inflamed atherosclerotic plaques of apolipoprotein E (apoE)-/- mice.
Materials and Methods
Galectins expression patterns in aortic atherosclerotic plaques and serum galectin-3 levels were investigated in 26-week-old apoE-/- (n=6) and C57BL/6 mice (n=9). To investigate the spatial and temporal patterns of galectin-1 and galectin-3 in plaques, high-cholesterol diet-fed 26-week-old (n=12) and 36-week-old apoE-/- mice (n=6) were sacrificed and their aortas were examined for galectins' expression using immunoblot analysis and immunohistochemical stain. 36-week-old apoE-/- mice were treated with atorvastatin (n=3, 0.57 mg/kg/day) for the evaluation of its effect on aortic galectins' expression.
Immunoblot analyses showed that galectin-1 and galectin-3 were the predominant galectins expressed in murine atherosclerosis. The serum galectin-3 level was significantly higher in apoE-/- mice (p<0.001). While galectin-1 was weakly expressed in both intimal plaques and the media of atherosclerotic aortas, galectin-3 was heavily and exclusively accumulated in intimal plaques. Galectin-3 distribution was colocalized with plaque macrophages' distribution (r=0.66). As the degree of plaque extent and inflammation increased, the intraplaque galectin-3 expression levels proportionally elevated (p<0.01 vs. baseline), whereas galectin-1 expression had not elevated (p=0.14 vs. baseline). Atorvastatin treatment markedly reduced intraplaque galectin-3 and macrophage signals (p<0.001 vs. baseline), whereas it failed to reduce galectin-1 expression in the aortas.
Galectin-3 is the predominant gal and is colocalized with macrophages within atherosclerotic plaques. Intraplaque galectin-3 expression reflects the degree of plaque inflammation.
PMCID: PMC3654109  PMID: 23682281
Galectin 1; Galectin 3; Atherosclerosis; Macrophages
18.  DPP-4 (CD26) Inhibitor Alogliptin Inhibits Atherosclerosis in Diabetic Apolipoprotein E-Deficient Mice 
Dipeptidyl peptidase-4 (DPP-4 or CD26) inhibitors, a new class of anti-diabetic compounds, are effective in treatment of hyperglycemia. Since atherosclerosis-related cardiovascular diseases are the major complications of diabetes, it is important to determine the effect of DPP-4 inhibitors on atherosclerosis. In this study, nondiabetic and diabetic apolipoprotein E (apoE)-deficient mice were treated with DPP-4 inhibitor alogliptin for 24 weeks and atherosclerotic lesions in aortic origins were examined. Results showed that diabetes significantly increased atherosclerotic lesions, but alogliptin treatment reduced atherosclerotic lesions in diabetic mice. Metabolic studies showed that diabetes increased plasma glucose and alogliptin treatment reduced glucose. Furthermore, immunohistochemistry study showed that diabetes increased IL-6 and IL-1β protein expression in atherosclerotic plaques, but alogliptin treatment attenuated diabetes-augmented IL-6 and IL-1β expression. In consistence with the observations from the mouse models, our in vitro studies showed that alogliptin inhibited toll-like receptor (TLR)4-mediated upregulation of IL-6, IL-1β, and other proinflammatory cytokines by mononuclear cells. Taken together, our findings showed that alogliptin inhibited atherosclerosis in diabetic apoE-deficient mice and the actions of alogliptin on both glucose and inflammation may contribute to the inhibition.
PMCID: PMC3155015  PMID: 21558879
Dipeptidyl peptidase-4; CD26; Atherosclerosis; Diabetes; Inflammation
19.  Enhanced Macrophage Tribbles-1 Expression in Murine Experimental Atherosclerosis 
Biology  2012;1(1):43-57.
Development of the atherosclerotic plaque involves a complex interplay between a number of cell types and an extensive inter-cellular communication via cell bound as well as soluble mediators. The family of tribbles proteins has recently been identified as novel controllers of pro-inflammatory signal transduction. The objective of this study was to address the expression pattern of all three tribbles proteins in atherosclerotic plaques from a mouse model of atherosclerosis. Each tribbles were expressed in vascular smooth muscle cells, endothelial cells as well as in resident macrophages of mouse atherosclerotic plaques. The role of IL-1 mediated inflammatory events in controlling tribbles expression was also addressed by inducing experimental atherosclerosis in ApoE−/−IL1R1−/− (double knockout) mice. Immunohistochemical analysis of these mice showed a selective decrease in the percentage of trb-1 expressing macrophages, compared to the ApoE−/− cohort (14.7% ± 1.55 vs. 26.3% ± 1.19). The biological significance of this finding was verified in vitro where overexpression of trb-1 in macrophages led to a significant attenuation (~70%) of IL-6 production as well as a suppressed IL-12 expression induced by a proinflammatory stimulus. In this in vitro setting, expression of truncated trb-1 mutants suggests that the kinase domain of this protein is sufficient to exert this inhibitory action.
PMCID: PMC4011034  PMID: 24832046
Tribbles; atherosclerosis; macrophages; mouse
20.  Association with inflammatory cells and apolipoproteins to the progression of atherosclerosis 
Inflammatory cells are known to be associated with the progression of atherosclerosis and plaque rupture. However, the relation to inflammatory cells and apolipoproteins on the progression of atherosclerosis is unknown. This study was aimed at examining the different expressions of inflammatory cells and evaluate the effect of apolipoprotein (APO) C1 and APO E during the progression of atherosclerosis.
Ten atherosclerotic tissues were compared with five non-atherosclerotic tissues. The presence of vascular smooth muscle cells (VSMCs), macrophages, T-cells, APO C1, and APO E were identified by Western blotting and immunohistochemical analysis with antibodies. The senescence was analyzed by senescence-associated β-galactosidase.
The protein expression and senescence of macrophages, APO C1 and APO E were significantly higher in the main atherosclerotic lesion than the non-atherosclerotic lesion. A high concentration of inflammatory cells and the paucity of VSMCs were present in the shoulder area. In addition, macrophage and T-cells are expressed in the early stage of atherosclerotic development and more expanded in advanced atherosclerotic plaques. APO C1 was expressed mainly within the necrotic core, and APO E existed mostly around the necrotic core and the fibrous cap in advanced atherosclerotic plaques.
Our study indicated that the expression and the senescence of macrophage and T-cells may be closelyrelated to induction and deposition of APO C1 and APO E. This contributes to the development and progression of atherosclerotic plaque by expanding the necrotic core.
PMCID: PMC3204681  PMID: 22066050
Atherosclerosis; Inflammatory cells; Apolipoproteins; Senescence
21.  Genetic reduction of lipoic acid synthase expression modestly increases atherosclerosis in male, but not in female, apolipoprotein E deficient mice 
Atherosclerosis  2010;211(2):424-430.
To evaluate the effects of a genetic reduction of Lias gene expression on atherosclerosis development.
Methods and Results
Heterozygous knockout mice for the lipoid acid synthase gene (Lias+/−) were crossed with apolipoprotein E–deficient (ApoE−/−) mice, and the plaque size in aortic sinuses of Lias+/− ApoE−/− mice was evaluated at 6 months of age. Lesions at the aortic sinus in Lias+/− ApoE−/− males were significantly larger (1.5X) than those in Lias+/+ ApoE−/− littermate males. The lesion size was inversely correlated with an increased erythrocyte reduced glutathione/ oxidized glutathione (GSH/GSSH) ratio, an systemic index of body redox balance. Lias+/− ApoE−/− males also had significantly increased plasma cholesterol and reduced pyruvate dehydrogenase complex activity in the liver. Significant reductions in the expression of genes for antioxidant enzymes, including superoxide dismutase 1 (SOD1) and SOD2, were observed in aortas of Lias+/− ApoE−/− males. Female Lias+/− ApoE−/− also exhibited changes in these parameters, parallel to those observed in males. However, the Lias gene effects for the majority of these factors, including atherosclerotic lesion size, were not significant in females.
Our data provide evidence that Lias deficiency enhances atherosclerosis in male mice, at least in part due to reduce antioxidant capacity. The notable absence of such effects in females leaves open the possibility of a gender-specific protection mechanism.
PMCID: PMC2914155  PMID: 20347443
antioxidant; lipoic acid; Lias mouse model; atherosclerosis; apolipoprotein E null mice
22.  Suppression of Atherogenesis by Overexpression of Glutathione Peroxidase-4 in Apolipoprotein E-Deficient Mice 
Free radical biology & medicine  2007;44(3):343-352.
Accumulation of oxidized lipids in the arterial wall contributes to atherosclerosis. Glutathione peroxidase-4 (GPx4) is a hydroperoxide scavenger that removes oxidative modifications from lipids such as free fatty acids, cholesterols, and phospholipids. Here, we set out to assess the effect of GPx4 overexpression on atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. The results revealed that atherosclerotic lesions in the aortic tree and aortic sinus of ApoE−/− mice overexpressing GPx4 (hGPx4Tg/ApoE−/−) were significantly smaller than those of ApoE−/− control mice. GPx4 overexpression also diminished signs of advanced lesions in the aortic sinus, as seen by a decreased occurrence of fibrous caps and acellular areas among hGPx4Tg/ApoE−/− animals. This delay of atherosclerosis in hGPx4Tg/ApoE−/− mice correlated with reduced aortic F2-isoprostane levels (R2 = 0.75, p < 0.01). In addition, overexpression of GPx4 lessened atherogenic events induced by the oxidized lipids, lysophosphatidylcholine and 7-ketocholesterol, including upregulated expression of adhesion molecules in endothelial cells, adhesion of monocytes to endothelial cells, as well as endothelial necrosis and apoptosis. These results suggest that overexpression of GPx4 inhibits the development of atherosclerosis by decreasing lipid peroxidation and inhibiting the sensitivity of vascular cells to oxidized lipids.
PMCID: PMC2245803  PMID: 18215741
Glutathione peroxidase-4; Atherosclerosis; Lipid peroxidation; Monocyte adhesion; Necrosis; Apoptosis
23.  Native incretins prevent the development of atherosclerotic lesions in apolipoprotein E knockout mice 
Diabetologia  2011;54(10):2649-2659.
Several lines of evidence suggest that incretin-based therapies suppress the development of cardiovascular disease in type 2 diabetes. We investigated the possibility that glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) can prevent the development of atherosclerosis in Apoe−/− mice.
Apoe−/− mice (17 weeks old) were administered GLP-1(7–36)amide, GLP-1(9–36)amide, GIP(1–42) or GIP(3–42) for 4 weeks. Aortic atherosclerosis, oxidised LDL-induced foam cell formation and related gene expression in exudate peritoneal macrophages were determined.
Administration of GLP-1(7–36)amide or GIP(1–42) significantly suppressed atherosclerotic lesions and macrophage infiltration in the aortic wall, compared with vehicle controls. These effects were cancelled by co-infusion with specific antagonists for GLP-1 and GIP receptors, namely exendin(9–39) or Pro3(GIP). The anti-atherosclerotic effects of GLP-1(7–36)amide and GIP(1–42) were associated with significant decreases in foam cell formation and downregulation of CD36 and acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in macrophages. GLP-1 and GIP receptors were both detected in Apoe−/− mouse macrophages. Ex vivo incubation of macrophages with GLP-1(7–36)amide or GIP(1–42) for 48 h significantly suppressed foam cell formation. This effect was wholly abolished in macrophages pretreated with exendin(9−39) or (Pro3)GIP, or with an adenylate cyclase inhibitor, MDL12,330A, and was mimicked by incubation with an adenylate cyclase activator, forskolin. The inactive forms, GLP-1(9–36)amide and GIP(3–42), had no effects on atherosclerosis and macrophage foam cell formation.
Our study is the first to demonstrate that active forms of GLP-1 and GIP exert anti-atherogenic effects by suppressing macrophage foam cell formation via their own receptors, followed by cAMP activation. Molecular mechanisms underlying these effects are associated with the downregulation of CD36 and ACAT-1 by incretins.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-011-2241-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC3168747  PMID: 21786155
Atherosclerosis; Cholesterol; Dipeptidyl peptidase-4; Glucagon-like peptide-1; Glucose-dependent insulinotropic polypeptide; Incretin; Macrophages; Mouse model
24.  Adhesive Strength of Atherosclerotic Plaque in a Mouse Model Depends on Local Collagen Content and Elastin Fragmentation 
Journal of biomechanics  2012;46(4):716-722.
Atherosclerotic plaque rupture is a major cause of myocardial infarction and ischemic stroke. The adhesive strength of the bond between a plaque and the vascular wall, measured as local energy release rate, G, is used for quantitative plaque stability estimation. We tested the hypothesis that adhesive strength varies with plaque composition. Matrix metalloproteinase-12 (MMP12) deficiency was previously reported to alter lesion composition. To estimate G values, peeling experiments are performed on aortic plaques from apolipoprotein E knockout (apoE KO) and apoE MMP12 double knockout (DKO) male mice after 8 months on high-fat diet. For plaques in apoE KO and apoE MMP12 DKO mice, experimental values for G differ significantly (p<0.002) between genotypes, averaging 19.2 Joule/m2 and 12.1 J/m2, respectively. Histology confirms that plaques delaminate along their interface with the underlying internal elastic lamina (IEL) in both genotypes. Quantitative image analysis of stained tissue sections demonstrates a significant positive correlation (p<0.05) between local collagen content of lesions and G values in both genotypes, indicating that adhesive strength of plaques depends on local collagen content. Surprisingly, macrophage content of aortic plaques is neither significantly correlated with G values nor significantly different between genotypes. The IEL underlying plaques in apoE KO mice is significantly more fragmented (number of breaks and length of breaks) than in apoE MMP12 DKO mice, suggesting that elastin fragmentation also influences adhesion strength of plaques. Overall, our results suggest that plaques adhere more strongly to the underlying IEL in apoE KO mice than in apoE MMP12 DKO mice.
PMCID: PMC3568211  PMID: 23261250
atherosclerosis; mouse models; energy release rate; plaque rupture; interfacial delamination; matrix metalloproteinase
25.  Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice 
PLoS ONE  2013;8(2):e57178.
Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoEnull) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoEnull mice.
PMCID: PMC3581444  PMID: 23451182

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