Low-density Lipoprotein Receptor-deficient Mice Research Articles

A novel thromboxane receptor antagonist and synthase inhibitor, BM-573, reduces development and progression of atherosclerosis in LDL receptor deficient mice

Atherosclerosis is a chronic inflammatory disease of the vasculature influenced by a variety of mediators. Among them, prostanoids, which include prostacyclin and thromboxane (Tx) A 2, have recently received a lot of attention. Previous studies demonstrated that antagonism or deletion of the receptor for TxA 2 retards early atherogenesis in apolipoprotein E-deficient mice, but no data are available in low-density lipoprotein (LDL) receptor deficient mice. In our study, we tested the effect of a novel TxA 2 receptor (TP) antagonist and synthase inhibitor, BM-573, on atherosclerosis development and progression in LDL receptor deficient mice. To this end, the effect of 12 weeks treatment with BM-573 on early or established aortic atherosclerotic lesions of these mice was assessed. In both treatments, while BM-573 did not affect body weight, systolic blood pressure, total plasma cholesterol or triglycerides levels, it partially reduced TxA 2 but did not affect prostacyclin biosynthesis. Moreover, BM-573 significantly decreased early atherogenesis and prevented progression of established atherosclerotic lesions. These results show for the first time that this dual Tx inhibitor is effective in reducing atherogenesis in the LDL receptor deficient mice. They also demonstrate the novel concept that this therapeutic approach halts the progression of the disease and influences the cellular composition of the atherosclerotic plaques.

Tesaglitazar, a dual peroxisome proliferator-activated receptor alpha/gamma agonist, reduces atherosclerosis in female low density lipoprotein receptor deficient mice

Objective The transcription factors, peroxisome proliferator-activated receptors (PPAR) alpha (α) and gamma (γ), which are involved in lipid and glucose homeostasis, also exert modulatory actions on vascular cells where they exhibit anti-inflammatory and anti-proliferative properties. Hence, PPAR agonists potentially can affect atherogenesis both via metabolic effects and direct effects on the vessel wall. We tested whether the dual PPAR-α/γ agonist, tesaglitazar (TZ), would reduce atherosclerosis in a non-diabetic, atherosclerosis-prone mouse model, independent of effects on plasma lipids. Methods and results Low-density lipoprotein receptor deficient (LDLr−/−) mice were fed a Western type diet consisting of 21% butterfat and 0.15% cholesterol, with or without TZ 0.5 μmol/kg of diet, for 12 weeks. TZ reduced atherosclerosis in the female, but not male, LDLr−/− mice without affecting cholesterol and triglyceride levels, HDL binding to biglycan, or the inflammatory markers serum amyloid A (SAA) and serum amyloid P (SAP). TZ also decreased adiposity in both genders. Conclusions TZ reduced atherosclerosis in the female LDLr−/− mice via lipid-independent mechanisms, probably at least in part by direct actions on the vessels. The body weight changes in these mice are different from the effects of dual PPAR agonists seen in humans.

Combined Treatment With Sustained-Release Basic Fibroblast Growth Factor and Heparin Enhances Neovascularization in Hypercholesterolemic Mouse Hindlimb Ischemia

Whether the combined treatment with sustained-release basic fibroblast growth factor (bFGF) and heparin enhances neovascularization in hypercholesterolemic mouse hindlimb ischemia was investigated. Wild-type C57BL/6 and low density lipoprotein receptor-deficient mice were assigned to 1 of the following 4 experimental groups and treated for 2 weeks after femoral artery extraction: group N, no treatment; group H, daily subcutaneous injection of heparin calcium; group F, single intramuscular injection of the sustained-release bFGF microspheres; and group FH, combined treatment with sustained-release bFGF and heparin. Among the wild-type mice at 4 weeks after femoral artery extraction, the laser Doppler perfusion image index (LDPII) in groups H, F, and FH was significantly higher than that in group N. The vascular density in group FH was the highest among the 4 groups. The maturation index in the 3 treated groups was significantly higher than that in group N. Among the hypercholesterolemic mice, the LDPII in group FH was significantly higher than that in the other 3 groups. The vascular density and maturation index in group FH were the highest among the 4 groups. Combined treatment with sustained-release bFGF and heparin enhanced neovascularization in the hypercholesterolemic hindlimb ischemia model.

Administration of Pioglitazone in Low-Density Lipoprotein Receptor-Deficient Mice Inhibits Lesion Progression and Matrix Metalloproteinase Expression in Advanced Atherosclerotic Plaques

Recent clinical trials have provided evidence that pioglitazone reduces cardiovascular events in patients with type 2 diabetes. However, the underlying mechanisms are not well understood. Because it has been well established that disruption of atherosclerotic plaques is a key event involved in acute myocardial infarction, we hypothesized that pioglitazone reduces cardiovascular events by stabilizing atherosclerotic lesions. In this study, we used an animal model to test our hypothesis. Low-density lipoprotein receptor-deficient (LDLR-/-) male mice were first fed a high-fat diet for 4 months to induce the formation of aortic atherosclerotic plaques and then treated with pioglitazone for the next 3 months. Analysis of atherosclerotic plaques at the end of the study showed that treatment with pioglitazone at 20 mg/kg/day reduced the progression of atherosclerotic plaques as compared to untreated mice. Furthermore, gene array analysis, quantitative real-time polymerase chain reaction, and immunohistochemical analysis showed that pioglitazone inhibited high-fat diet-induced upregulation of matrix metalloproteinase (MMP) expression. Finally, Sirius red staining showed that atherosclerotic lesions in mice receiving pioglitazone had higher collagen contents than those in untreated mice. This study demonstrated for the first time that administration of pioglitazone in LDLR-/- mice inhibited lesion progression and MMP expression in established atherosclerotic plaques and thus delineated a potential mechanism by which pioglitazone reduces cardiovascular events in patients with type 2 diabetes.

Interruption of the Tnfrsf4/Tnfsf4 (OX40/OX40L) Pathway Attenuates Atherogenesis in Low-Density Lipoprotein Receptor-Deficient Mice

Atherosclerosis is a chronic (auto-)inflammatory disease and T cell activation is an important factor in this process. Tnfrsf4 (OX40) and Tnfsf4 (OX40 ligand) are members of the tumor necrosis factor (TNF) and TNF receptor family and OX40/OX40L mediated signaling is important in co-activation of T cells and facilitates B-T cell interaction. In this study we assessed the role of the OX40/OX40L pathway in atherosclerosis and the effect of interruption of the OX40/OX40L pathway on lesion development. We treated low-density lipoprotein receptor-deficient (LDLr-/-) mice with an anti-OX40L antibody which lead to a 53% decrease in atherosclerotic lesion formation. Treatment resulted in inhibition of Th2 mediated isotype switching by decreasing interleukin (IL)-4 secretion and subsequent low IgG1 serum levels against oxLDL, whereas protective anti-oxLDL specific IgM titers were increased in treated mice compared with control. We conclude that blocking the OX-40/OX40L interaction reduced atherogenesis by inhibition of IL-4 mediated Th2 induced isotype switching and subsequent increased levels of anti-oxLDL IgM.

Short-Term Treatment With Anti-CD3 Antibody Reduces the Development and Progression of Atherosclerosis in Mice

Atherosclerosis is a chronic inflammatory disease of the large arteries that is the primary cause of heart disease and stroke. Anti-CD3-specific antibodies suppress immune responses by antigenic modulation of the CD3 antibody/T-cell receptor complex. Their unique capacity to restore self-tolerance in a mouse model of diabetes and, importantly, in patients with recent-onset type 1 diabetes involves transforming growth factor-beta-dependent mechanisms via expansion and/or activation of regulatory T cells. We hypothesized that treatment with anti-CD3-specific antibodies might inhibit atherosclerosis development and progression in mice. Low-density lipoprotein receptor-deficient mice were fed a high-cholesterol diet for 13 or 24 weeks. Anti-CD3 antibody was administered on 5 consecutive days beginning 1 week before or 13 weeks after the high-cholesterol diet was initiated, respectively. Control mice were injected in parallel with phosphate-buffered saline. Anti-CD3 antibody therapy reduced plaque development when administered before a high-cholesterol diet and markedly decreased lesion progression in mice with already established atherosclerosis. We found increased production of the antiinflammatory cytokine transforming growth factor-beta in concanavalin A-stimulated lymph node cells and enhanced expression of the regulatory T-cell marker Foxp3 in spleens of anti-CD3 antibody-treated mice. A higher percentage of apoptotic cells within the plaques of anti-CD3 antibody-treated mice was also observed. Altered disease progression, combined with the emergence of this particular cytokine pattern, indicates that short-term treatment with an anti-CD3 antibody induces a regulatory T-cell phenotype that restores self-tolerance in a mouse model of atherosclerosis.

Macrophage ABCG1 Deletion Disrupts Lipid Homeostasis in Alveolar Macrophages and Moderately Influences Atherosclerotic Lesion Development in LDL Receptor-Deficient Mice

ABCG1 has recently been identified as a facilitator of cellular cholesterol and phospholipid efflux to high-density lipoprotein (HDL). Its expression in macrophages is induced during cholesterol uptake in macrophages and by liver X receptor (LXR). The role of macrophage ABCG1 in atherosclerotic lesion development is, however, still unknown. To assess the role of macrophage ABCG1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr-/-) mice that are selectively deficient in macrophage ABCG1 by using bone marrow transfer (ABCG1-/- --> LDLr-/-). Peritoneal macrophages isolated from donor ABCG1-/- mice exhibited a 22% (P=0.0007) decrease in cholesterol efflux to HDL. To induce atherosclerosis, transplanted mice were fed a high-cholesterol diet containing 0.25% cholesterol and 15% fat for 6 and 12 weeks. Serum lipid levels and lipoprotein profiles did not differ significantly between ABCG1-/- --> LDLr-/- mice and controls. In lungs of ABCG1-/- --> LDLr-/- mice a striking accumulation of lipids was observed in macrophages localized to the subpleural region. After 6 weeks of high-cholesterol diet feeding the atherosclerotic lesion size was 49+/-12x10(3) microm2 for ABCG1+/+ --> LDLr-/- mice versus 65+/-15x103 microm2 for ABCG1-/- --> LDLr-/- mice and after 12 weeks of high-cholesterol diet feeding 124+/-17x10(3) microm2 for ABCG1+/+ --> LDLr-/- mice versus 168+/-17x10(3) microm2 for ABCG1-/- --> LDLr-/- mice. Atherosclerotic lesion size depended on both time and the macrophage ABCG1 genotype (P=0.038 by 2-way ANOVA, n > or = 8), indicating a moderately 33% to 36% increase in lesion formation in the absence of macrophage ABCG1. Macrophage ABCG1 deficiency does lead to heavy lipid accumulation in macrophages of the lung, and also a moderately significant effect on atherosclerotic lesion development was observed.

Addition of dietary fat to cholesterol in the diets of LDL receptor knockout mice: effects on plasma insulin, lipoproteins, and atherosclerosis

The factors underlying cardiovascular risk in patients with diabetes have not been clearly elucidated. Efforts to study this in mice have been hindered because the usual atherogenic diets that contain fat and cholesterol also lead to obesity and insulin resistance. We compared plasma glucose, insulin, and atherosclerotic lesion formation in LDL receptor knockout (Ldlr(-/-)) mice fed diets with varying fat and cholesterol content that induced similar lipoprotein profiles. Ldlr(-/-) mice fed a high-fat diet developed obesity, mild hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. Quantitative and qualitative assessments of atherosclerosis were unchanged in diabetic Ldlr(-/-) mice fed a high-fat diet compared with lean nondiabetic control mice after 20 weeks of diet. Although one group of mice fed diets for 40 weeks had larger lesions at the aortic root, this was associated with a more atherogenic lipoprotein profile. The presence of a human aldose reductase transgene had no effect on atherosclerosis in fat-fed Ldlr(-/-) mice with mild diabetes. Our data suggest that when lipoprotein profiles are similar, addition of fat to a cholesterol-rich diet does not increase atherosclerotic lesion formation in Ldlr(-/-) mice.

LipoCardium: Endothelium-directed cyclopentenone prostaglandin-based liposome formulation that completely reverses atherosclerotic lesions

Atherosclerosis is a multifactorial inflammatory disease of blood vessels which decimates one in every three people in industrialized world. Despite the important newest clinical approaches, currently available strategies (e.g. nutritional, pharmacological and surgical) may only restrain the worsening of vascular disease. Since antiproliferative cyclopentenone prostaglandins (CP-PGs) are powerful anti-inflammatory agents, we developed a negatively charged liposome-based pharmaceutical formulation (LipoCardium) that specifically direct CP-PGs towards the injured arterial wall cells of atherosclerotic mice. In the blood stream, LipoCardium delivers its CP-PG contents only into activated arterial wall lining cells due to the presence of antibodies raised against vascular cell adhesion molecule-1 (VCAM-1), which is strongly expressed upon inflammation by endothelial cells and macrophage-foam cells as well. After 4 months in a high-lipid diet, all low-density lipoprotein receptor-deficient adult control mice died from myocardium infarction or stroke in less than 2 weeks, whereas LipoCardium-treated (2 weeks) animals (still under high-lipid diet) completely recovered from vascular injuries. In vitro studies using macrophage-foam cells suggested a tetravalent pattern for LipoCardium action: anti-inflammatory, antiproliferative (and pro-apoptotic only to foam cells), antilipogenic and cytoprotector (via heat-shock protein induction). These astonishing cellular effects were accompanied by a marked reduction in arterial wall thickness, neointimal hyperplasia and lipid accumulation, while guaranteed lifespan to be extended to the elderly age. Our findings suggest that LipoCardium may be safely tested in humans in a near future and may have conceptual implications in atherosclerosis therapy.

Pioglitazone inhibits connective tissue growth factor expression in advanced atherosclerotic plaques in low-density lipoprotein receptor-deficient mice

Connective tissue growth factor (CTGF) is expressed in atherosclerotic plaques. It is generally recognized that CTGF contributes to atherosclerosis by stimulating vascular smooth muscle cell (VSMC) proliferation and extracellular matrix production during the development of atherosclerosis. Recent studies indicate that CTGF may also contribute to plaque destabilization as it induces apoptosis and stimulates MMP-2 expression in VSMCs. Thiazolidinediones (TZDs), a new class of insulin sensitizing drugs for type 2 diabetes, inhibit atherosclerosis. However, their effect on CTGF expression in atherosclerotic plaques remains unknown. In this study, male LDL receptor-deficient mice were fed high-fat diet for 4 months to induce the formation of atherosclerotic plaques and then given the high-fat diet with or without pioglitazone for the next 3 months. At the end of the 7-month study, CTGF expression in aortic atherosclerotic lesions was examined. Results showed that CTGF expression was increased in mice fed the high-fat diet by seven-fold as compared to that in mice fed normal chow, but the treatment with pioglitazone significantly inhibited the high-fat diet-induced CTGF expression. To verify these in vivo observations, in vitro studies using human aortic SMC were conducted. Quantitative real-time PCR and Western blot showed that pioglitazone inhibited TGF-β-stimulated CTGF expression. In conclusion, the present study has demonstrated that pioglitazone inhibits CTGF expression in mouse advanced atherosclerotic plaques and in cultured human SMCs, and hence unveiled a possible mechanism potentially involved in the inhibition of atherosclerosis by TZD.

Differential effects of apolipoprotein E isoforms on lipolysis of very low-density lipoprotein triglycerides

Apolipoprotein (apo) E plays a key role in lipoprotein metabolism and has been proposed to modulate triglyceride (TG) lipolysis. However, no systematic investigation on lipolysis using all 3 isoforms of apoE has been performed. To clarify the role of common human apoE isoforms in the lipolysis of very low-density lipoprotein (VLDL) TGs, we overexpressed human apoE isoforms in apoE and low-density lipoprotein receptor–deficient mice using adenoviral-mediated gene transfer and used VLDL particles obtained from these mice for in vitro lipolysis assay. Overexpression of apoE, regardless of its isoforms, increased the TG content of VLDL in mice in vivo. In vitro analysis of the effect of apoE on lipolysis revealed that irrespective of its isoforms, apoE did inhibit TG lipolysis at every concentration of apoE examined, and this inhibitory effect became more pronounced as the apoE content of VLDL increased. No difference was observed in TG lipolysis activity among isoforms at low apoE/TG ratio; however, intermediate ratios of apoE/TG, which reflect physiologic VLDL apoE/TG ratios, demonstrated a significantly greater level of lipolysis inhibition in apoE2, but less so in apoE4 compared with other isoforms. This differential effect by apoE isoforms on lipolysis was attenuated at higher apoE/TG ratios; nevertheless, apoE2 still inhibited lipolysis significantly more than did apoE4. Enrichment of VLDL with apoE decreased both the apoC contents and apoC-II/C-III ratios of VLDL, contributing, at least in part, to the inhibitory function of apoE on lipolysis. The present study clarifies the differential lipolysis-modulating effect of apoE isoforms, which would help explain the difference in pre- and postprandial TG levels among humans carrying different apoE isoforms.

Apolipoprotein M – a novel player in high-density lipoprotein metabolism and atherosclerosis

Apolipoprotein M is a recently described apolipoprotein predominantly associated with high-density lipoprotein, but also found in chylomicrons, very low-density lipoproteins, and low-density lipoprotein. The purpose is to review recent information on the unusual structural properties of apolipoprotein M and its possible role in formation of pre-beta high-density lipoprotein and reverse cholesterol metabolism. Apolipoprotein M is a lipocalin having a coffee filter-like structure with a hydrophobic ligand-binding pocket. Mature apolipoprotein M retains its signal peptide, which serves as a hydrophobic anchor. In mice, silencing of expression in the liver with siRNA led to disappearance of pre-beta high-density lipoprotein and appearance of unusually large high-density lipoproteins. This suggests that apolipoprotein M is important for the formation of pre-beta high-density lipoprotein and reverse cholesterol transport. In accordance with this idea, hepatic overexpression of apolipoprotein M with an adenovirus in low-density lipoprotein-receptor deficient mice led to an approximately 70% reduction of atherosclerosis. In addition to the liver, apolipoprotein M is also expressed in the kidney. Kidney-derived apolipoprotein M binds to megalin, a member of the low-density lipoprotein-receptor family, which interacts with many lipocalins in renal tubuli. Apolipoprotein M is excreted in the urine of mice with a kidney-specific megalin deficiency but not in the urine of normal mice, suggesting megalin-mediated uptake of apolipoprotein M in the tubular epithelium of normal mice. Apolipoprotein M is a novel apolipoprotein with unusual structural features that appears to play important roles in high-density lipoprotein metabolism and prevention of atherosclerosis.

Atheroprotective Potential of Macrophage-Derived Phospholipid Transfer Protein in Low-Density Lipoprotein Receptor-Deficient Mice Is Overcome by Apolipoprotein AI Overexpression

Using bone marrow transplantation, we assessed the impact of macrophage-derived phospholipid transfer protein (PLTP) on lesion development in hypercholesterolemic mice that expressed either normal levels of mouse apolipoprotein AI (apoAI) or elevated levels of only human apoAI. Bone marrow transplantations were performed in low-density lipoprotein receptor-deficient mice (LDLr-/-) that expressed either normal levels of mouse apoAI (msapoAI) or high levels of only human apoAI (msapoAI-/-, LDLr-/-, huapoAITg). Mice were lethally irradiated, reconstituted with either PLTP-expressing or PLTP-deficient bone marrow cells, and fed a high-fat diet over 16 weeks. Macrophage PLTP deficiency increased atherosclerosis in LDLr-/- mice with minimal changes in total plasma cholesterol levels. In contrast, the extent of atherosclerosis in msapoAI-/-, LDLr-/-, huapoAITg mice was not significantly different between groups that had received PLTP-/- or PLTP+/+ bone marrow. In vitro studies indicated that PLTP deficiency led to a significant decrease in alpha-tocopherol content and increased oxidative stress in bone marrow cells. Our observations suggest an atheroprotective role of macrophage-derived PLTP in mice with normal apoAI plasma levels. The atheroprotective properties of macrophage-derived PLTP were not observable in the presence of elevated plasma concentrations of apoAI.

Immune dysregulation accelerates atherosclerosis and modulates plaque composition in systemic lupus erythematosus

Patients with systemic lupus erythematosus (SLE) have accelerated atherosclerosis. The underlying mechanisms are poorly understood, and investigations have been hampered by the absence of animal models that reflect the human condition of generalized atherosclerosis and lupus. We addressed this problem by transferring lupus susceptibility to low-density lipoprotein (LDL) receptor-deficient (LDLr-/-) mice, an established model of atherosclerosis, creating radiation chimeras with NZM2410-derived, lupus-susceptible, B6.Sle1.2.3 congenic or C57BL/6 control donors (LDLr.Sle and LDLr.B6, respectively). LDLr.Sle mice developed a lupus-like disease characterized by production of double-stranded DNA autoantibodies and renal disease. When fed a Western-type diet, LDLr.Sle chimeras had increased mortality and atherosclerotic lesions. The plaques of LDLr.Sle mice were highly inflammatory and contained more CD3+ T cells than controls. LDLr.Sle mice also had increased activation of CD4+ T and B cells and significantly higher antibody to oxidized LDL and cardiolipin. Collectively, these studies demonstrate that the lupus-susceptible immune system enhances atherogenesis and modulates plaque composition.

A mouse monoclonal antibody specific for mouse apoB48 and apoB100 produced by immunizing “apoB39-only” mice with mouse apoB48

We have generated and characterized a murine monoclonal antibody (mAb) that binds to both mouse apolipoprotein (apo) B48 and apoB100. We immunized “apoB39-only” mice (mice that synthesize a truncated form of apoB, apoB39, but no apoB48 or apoB100) with lipoproteins containing mouse apoB48 and then used splenocytes from the immunized mice to create hybridomas. We identified a hybridoma, 2G11, that secretes a mAb that binds to mouse apoB48 and apoB100 but not to apoB39. Antibody 2G11 also binds apoB48 and apoB100 from rats and hamsters but not from humans. The mAb recognizes mouse apoB equally in very low and low density lipoproteins and was used to quantify apoB in wild-type, apoE-deficient and low-density lipoprotein receptor-deficient mice and in mice treated with an antisense drug that lowers plasma apoB levels. The antibody will be an important reagent for studying mouse models of atherosclerosis. The study also underscores the utility of genetically modified mice for generating mouse mAbs against mouse proteins.

A High Fat/High Carbohydrate Diet Induces Aortic Valve Disease in C57BL/6J Mice

The purpose of this study was to compare aortic valve function and morphology in adult wild-type (WT) mice and in low-density lipoprotein receptor-deficient (LDLr-/-) mice fed or not fed a high-fat/high-carbohydrate (HF/HC) diet. Observations suggest a link between degenerative aortic valve stenosis (AS) and atherosclerosis. Aortic valve stenosis has been successfully induced in animal models of extreme hypercholesterolemia, but these models are less relevant to humans. It is not known if a proatherogenic HF/HC diet without added cholesterol could have the same negative impacts. Forty C57BL/6J mice were divided into four groups: WT + normal diet, WT + HF/HC diet, LDLr-/- with a normal diet, and LDLr-/- with a HF/HC diet. Aortic valve function and histology were evaluated by echocardiography after four months. Wild-type mice on a HF/HC diet became mildly hypercholesterolemic, obese, and hyperglycemic. As expected, LDLr-/- mice became severely hypercholesterolemic. Both WT and LDLr-/- mice on a HF/HC diet displayed smaller valve areas and higher transvalvular velocities (p < 0.01) after four months. Aortic valve leaflets were thicker and infiltrated with lipids and macrophages in both HF/HC groups. A HF/HC diet in mice results in significant aortic valve abnormalities. Putting WT mice on a HF/HC diet reproduced a combination of atherogenic factors (obesity, mild dyslipidemia, and hyperglycemia) more commonly encountered in humans than isolated severe hypercholesterolemia. Severe hypercholesterolemia was not a prerequisite in our model. This experimental model suggests that AS development is multifactorial and that hypercholesterolemia should not be the only target in this disease.

Atherosclerosis in Mice Is Not Affected by a Reduction in Tissue Factor Expression

To determine whether tissue factor (TF) contributes to the progression of atherosclerotic lesions in mice. We determined the effect of a 50% reduction of TF levels in all cells on atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. No differences were observed in the extent of atherosclerosis in apoE(-/-)/TF(+/+) and apoE(-/-)/TF(+/-) mice fed regular chow for 34 weeks. Atherosclerosis could not be analyzed in apoE(-/-) mice expressing low levels of TF because of premature death of these mice. Macrophages are a major source of TF in atherosclerotic plaques. Therefore, in a second series of experiments, we investigated the effect on atherosclerosis of selectively reducing hematopoietic cell-derived TF by transplanting bone marrow from mice expressing low levels of TF into low-density lipoprotein receptor deficient (LDLR(-/-)) mice. Atherosclerosis within the arterial tree and aortic root were similar in LDLR(-/-) mice with low-TF bone marrow compared with control bone marrow (TF(+/+) or TF(+/-)) after 4 and 16 weeks on an atherogenic diet. Furthermore, the cellular composition of the aortic root lesions was similar between the 2 groups. Our data indicate that either a 50% reduction of TF in all cells or a selective reduction in hematopoietic cell-derived TF does not affect the development of atherosclerotic lesions in mice.

The role of dietary oxidized cholesterol and oxidized fatty acids in the development of atherosclerosis

The etiology of atherosclerosis is complex and multifactorial but there is extensive evidence indicating that oxidized lipoproteins may play a key role. At present, the site and mechanism by which lipoproteins are oxidized are not resolved, and it is not clear if oxidized lipoproteins form locally in the artery wall and/or are sequestered in atherosclerotic lesions following the uptake of circulating oxidized lipoproteins. We have been focusing our studies on demonstrating that such potentially atherogenic oxidized lipoproteins in the circulation are at least partially derived from oxidized lipids in the diet. Thus, the purpose of our work has been to determine in humans whether oxidized dietary oxidized fats such as oxidized fatty acids and oxidized cholesterol are absorbed and contribute to the pool of oxidized lipids in circulating lipoproteins. When a meal containing oxidized linoleic acid was fed to normal subjects, oxidized fatty acids were found only in the postprandial chylomicron/chylomicron remnants (CM/RM) which were cleared from circulation within 8 h. No oxidized fatty acids were detected in low density lipoprotein (LDL) or high density lipoprotein (HDL) fractions at any time. However, when alpha-epoxy cholesterol was fed to human subjects, alpha-epoxy cholesterol in serum was found in CM/RM and also in endogenous very low density lipoprotein, LDL, and HDL and remained in the circulation for 72 h. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. We have suggested that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into CM/RM fraction and then transferred to LDL and HDL contributing to lipoprotein oxidation. We hypothesize that diet-derived oxidized fatty acids in chylomicron remnants and oxidized cholesterol in remnants and LDL accelerate atherosclerosis by increasing oxidized lipid levels in circulating LDL and chylomicron remnants. This hypothesis is supported by our feeding experiments in animals. When rabbits were fed oxidized fatty acids or oxidized cholesterol, the fatty streak lesions in the aorta were increased by 100%. Moreover, dietary oxidized cholesterol significantly increased aortic lesions in apo-E and LDL receptor-deficient mice. A typical Western diet is rich in oxidized fats and therefore could contribute to the increased arterial atherosclerosis in our population.

HIV Entry Inhibitor TAK-779 Attenuates Atherogenesis in Low-Density Lipoprotein Receptor–Deficient Mice

HIV combination therapy using protease inhibitors is associated with elevated plasma levels of atherogenic lipoproteins and increased risk for atherosclerosis. We investigated whether the HIV entry inhibitor TAK-779 affects lipoprotein levels and atherogenesis in low-density lipoprotein receptor-deficient mice. TAK-779 is an antagonist for the chemokine receptors CCR5 and CXCR3, which are expressed on leukocytes, especially T-helper 1 cells, and these receptors may be involved in recruitment of these cells to atherosclerotic plaques. TAK-779 treatment of low-density lipoprotein receptor-deficient mice did not elevate the levels of atherogenic lipoproteins, whereas it dramatically reduced atherosclerosis in the aortic root and in the carotid arteries. The number of T cells in the plaque was reduced by 95%, concurrently with a 98% reduction in the relative IFN-gamma area. TAK-779-treated animals showed a decreased percentage of CD4+ and CD8+ T cells in peripheral blood and in mediastinal lymph nodes compared with control-treated animals. TAK-779 not only suppresses HIV entry via blockade of CCR5 but also attenuates atherosclerotic lesion formation by blocking the influx of T-helper 1 cells into the plaque. TAK-779 treatment may be especially beneficial for young HIV patients as they face lifelong treatment, and this drug impairs atherogenesis.

Modulation of atherosclerosis in mice by Toll-like receptor 2

Epidemiologic evidence has established a relationship between microbial infection and atherosclerosis. Mammalian TLRs provide clues on the mechanism of this inflammatory cascade. TLR2 has a large ligand repertoire that includes bacterial-derived exogenous and possibly host-derived endogenous ligands. In atherosclerosis-susceptible low-density lipoprotein receptor-deficient (Ldlr-/-) mice, complete deficiency of TLR2 led to a reduction in atherosclerosis. However, with BM transplantation, loss of TLR2 expression from BM-derived cells had no effect on disease progression. This suggested that an unknown endogenous TLR2 agonist influenced lesion progression by activating TLR2 in cells that were not of BM cell origin. Moreover, with intraperitoneal administration of a synthetic TLR2/TLR1 agonist, Pam3CSK4, disease burden was dramatically increased in Ldlr-/- mice. A complete deficiency of TLR2 in Ldlr-/- mice, as well as a deficiency of TLR2 only in BM-derived cells in Ldlr-/- mice, led to striking protection against Pam3CSK4-mediated atherosclerosis, suggesting a role for BM-derived cell expression of TLR2 in transducing the effects of an exogenous TLR2 agonist. These studies support the concept that chronic or recurrent microbial infections may contribute to atherosclerotic disease. Additionally, these data suggest the presence of host-derived endogenous TLR2 agonists.