Bone marrow-derived multidrug resistance protein ABCB4 protects against atherosclerotic lesion development in LDL receptor knockout mice

OBJECTIVE: Several members of the ATP binding cassette (ABC)-transporter super family expressed in macrophages protect against atherosclerosis by promoting macrophage cholesterol and phospholipid efflux. Systemic disruption of ABCB4 in mice results in a virtual absence of phospholipids in bile and a strongly impaired biliary cholesterol secretion, indicating that ABCB4 plays an essential role in cellular lipid efflux. The aim of the current study was to determine the role of bone marrow-derived ABCB4 in atherosclerotic lesion development. METHODS: Chimeras were created that specifically lack ABCB4 in bone marrow-derived cells, including macrophages, by performing a bone marrow transplantation on LDL receptor knockout (LDLr-/-) mice. Atherosclerotic lesion development was induced by feeding a high-cholesterol diet (15% fat and 0.25% cholesterol). RESULTS: Serum cholesterol levels were significantly lower in mice reconstituted with ABCB4 knockout bone marrow as a result of reduced VLDL and LDL cholesterol levels. Despite the lower serum cholesterol levels, ABCB4 deficiency in bone marrow-derived cells resulted in a 1.8-fold (p=0.005) increase in lesion size. In vitro foam cell formation, induced with acetylated LDL (AcLDL) in peritoneal macrophages, was increased in the absence of ABCB4, possibly due to a 2-fold (p<0.05) increased association of AcLDL, while the efflux of cholesterol was unaffected. CONCLUSION: Bone marrow-derived ABCB4 has an important anti-atherosclerotic function, probably by limiting macrophage foam cell formation.     

Important role for bone marrow-derived cholesteryl ester transfer protein in lipoprotein cholesterol redistribution and atherosclerotic lesion development in LDL receptor knockout mice

Abundant amounts of cholesteryl ester transfer protein (CETP) are found in macrophage-derived foam cells in the arterial wall, but its function in atherogenesis is unknown. To investigate the role of macrophage CETP in atherosclerosis, LDL receptor knockout mice were transplanted with bone marrow from CETP transgenic mice, which express the human CETP transgene under control of its natural promoter and major regulatory elements. CETP production by bone marrow-derived cells induced a 1.8-fold (P<0.01) increase in atherosclerotic lesion development. The increase in lesion size coincided with an increase in VLDL/LDL cholesterol and a decrease in HDL cholesterol. The cholesterol redistribution in serum was a direct effect of the substantial serum CETP activity and mass (38+/-3 nmol/mL/h and 4.8+/-0.5 microg/mL, respectively) induced by CETP production by bone marrow-derived cells. Conversely, specific disruption of CETP production by bone marrow-derived cells in CETP transgenic mice resulted in a approximately 2-fold (P<0.0001) reduction in serum CETP activity and mass, demonstrating the quantitative relevance of bone marrow-derived CETP. Finally, we show that in liver Kupffer cells, hepatic macrophages, contribute approximately 50% to the total hepatic CETP expression. In conclusion, bone marrow-derived CETP induces a proatherogenic lipoprotein profile and promotes the development of atherosclerotic lesions in LDL receptor knockout mice. Most importantly, we show for the first time that bone marrow-derived CETP is an important contributor to total serum CETP activity and mass.     

Selective effect of conjugated linoleic acid isomers on atherosclerotic lesion development in apolipoprotein E knockout mice

Research suggests that conjugated linoleic acid (CLA) may inhibit atherosclerosis, but there are contradictory results in different animal models fed heterogeneous mixtures of CLA isomers. This study addressed the hypothesis that the individual CLA isomers may exert different atherogenic properties. ApoE(-/-) mice were fed isocaloric, isonitrogenous westernized diets containing 0.15% cholesterol and enriched with 1% (w/w) cis-9,trans-11-CLA (c9,t11-CLA), trans-10,cis-12-CLA (t10,c12-CLA) or linoleic acid (control diet) for 12 weeks. At the end of the dietary intervention, the effects of CLA isomers on the development of atherosclerotic vascular lesions, lipid metabolism, inflammation and oxidative stress were assessed. The t10,c12-CLA diet had a profound pro-atherogenic effect, whereas c9,t11-CLA impeded the development of atherosclerosis. En face aortic lesion assessment showed more dorsal and lumbar extensions presenting atherosclerotic foci after the t10,c12-CLA diet. Furthermore, animals fed t10,c12-CLA had pronounced hyperlipidemia, higher 8-iso-prostaglandin F(2alpha) levels, higher vulnerable atherosclerotic plaque with a lower smooth muscle and fibre contents and higher macrophage content and activation, assayed as plasma chitotriosidase compared to the control or c9,t11-CLA dietary groups. Plasma chitotriosidase activity was more closely associated with the extent of the plaque than with MOMA staining or than monocyte chemoattractant protein-1 levels. Our results demonstrate that CLA isomers differentially modulate the development of atherosclerosis, c9,t11-CLA impedes, whereas t10,c12-CLA promotes atherosclerosis. These opposing effects may be ascribed to divergent effects on lipid, oxidative, inflammatory and fibro muscular components of this pathology. Plasma chitotriosidase is a better indicator of dietary fat interventions that alter plaque monocyte activity in this murine model.     

Heme oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout mice

Heme oxygenase-1 (HO-1) is induced by a variety of conditions associated with oxidative stress. We demonstrated that mildly oxidized LDL markedly induces HO-1 in human aortic endothelial and smooth muscle cell cocultures and that its induction results in the attenuation of monocyte chemotaxis resulting from treatment with mildly oxidized LDL in vitro. To elucidate the role of HO-1 in the development of atherosclerotic lesions in vivo, we modulated HO-1 expression in LDL-receptor knockout mice fed high-fat diets. During 6-week high-fat diet trials, intraperitoneal injections of hemin (H group) or hemin and desferrioxamine (HD group) to induce HO-1, Sn-protoporphyrin IX to inhibit HO-1 (Sn group), and saline as control (C group) were performed. Both the H and HD groups showed significantly less mean atherosclerotic lesions in the proximal aorta compared with the C group, whereas the Sn group showed larger lesion compared with the C group. Modulation of HO expression and HO activities were confirmed by Northern blot analysis and HO activity assay. Immunohistochemical studies revealed significant HO-1 expression in atherosclerotic lesions, where oxidized phospholipids also localized. Major cell types expressing HO-1 were macrophages and foam cells in the lesions. HO modulations affected plasma lipid hydroperoxide (LPO) levels and nitrite/nitrate levels. These results suggest that HO-1, induced under hyperlipidemia, functioned as an intrinsic protective factor against atherosclerotic lesion formation, possibly by inhibiting lipid peroxidation and influencing the nitric oxide pathway.     

Interleukin-4 deficiency decreases atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/- mice

Activated lymphocytes and mast cells have been detected in human atherosclerotic lesions. Interleukin-4 (IL-4) is a prominent cytokine released during the activation of both these cell types, and its mRNA has been detected in human and mouse atherosclerotic lesions. To define the effects of IL-4 on atherogenesis, bone marrow stem cells from either IL-4-/- or IL-4+/+ mice were transplanted into lethally irradiated female low density lipoprotein (LDL) receptor-/- mice. After an interval sufficient to allow engraftment, mice were placed on a diet containing 21% saturated fat, 1.25% cholesterol, and 0.5% cholate. Hematopoietic engraftment was confirmed by the presence of the LDL receptor gene in bone marrow cells. The effect on IL-4 depletion was confirmed by quantifying cytokine release from splenocytes of reconstituted mice. The deficiency of IL-4 in bone marrow-derived cells had no effect on serum cholesterol concentrations or on the distribution of cholesterol among lipoproteins. Atherosclerotic lesion formation was not changed in the aortic root. However, deficiency of IL-4 led to reduced lesion size in the arch (9.1 +/- 1.1% versus 2.8 +/- 0.8% of intimal area, P<0.001) and the thoracic aorta (1.2 +/- 0.2% versus 0.4 +/- 0.1%, P<0.002). Therefore, IL-4 deficiency reduced atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/- mice fed a high-fat diet.     

Myeloid-specific estrogen receptor alpha deficiency impairs metabolic homeostasis and accelerates atherosclerotic lesion development

ERα is expressed in macrophages and other immune cells known to exert dramatic effects on glucose homeostasis. We investigated the impact of ERα expression on macrophage function to determine whether hematopoietic or myeloid-specific ERα deletion manifests obesity-induced insulin resistance in mice. Indeed, altered plasma adipokine and cytokine levels, glucose intolerance, insulin resistance, and increased adipose tissue mass were observed in animals harboring a hematopoietic or myeloid-specific deletion of ERα. A similar obese phenotype and increased atherosclerotic lesion area was displayed in LDL receptor-KO mice transplanted with ERα(-/-) bone marrow. In isolated macrophages, ERα was necessary for repression of inflammation, maintenance of oxidative metabolism, IL-4-mediated induction of alternative activation, full phagocytic capacity in response to LPS, and oxidized LDL-induced expression of ApoE and Abca1. Furthermore, we identified ERα as a direct regulator of macrophage transglutaminase 2 expression, a multifunctional atheroprotective enzyme. Our findings suggest that diminished ERα expression in hematopoietic/myeloid cells promotes aspects of the metabolic syndrome and accelerates atherosclerosis in female mice.     

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice

The cyclooxygenase (COX) product, prostacyclin (PGI(2)), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI(2) biosynthesis substantially in humans. Because deletion of the PGI(2) receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF(1alpha) by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI(2) biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 +/- 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.     

CCR5 deficiency is not protective in the early stages of atherogenesis in apoE knockout mice

The accumulation of macrophages and T lymphocytes in vessel walls is a hallmark of atherogenesis. It has recently been demonstrated in mouse models of atherosclerosis that full disease potential is dependent on several regulators of leukocyte trafficking, including the chemokine monocyte chemotactic protein 1 (MCP-1) and the chemokine receptors CCR2 and CXCR2. A possible role for the chemokine receptor CCR5 in atherogenesis has been suggested by CCR5 expression on macrophages, T cells, coronary endothelial cells and aortic smooth muscle cells and by the presence of CCR5 ligands in atherosclerotic plaques. Moreover, individuals who are naturally deficient in CCR5 were reported to be at reduced risk for severe coronary artery disease (CAD) and early myocardial infarction (MI). To investigate whether CCR5 is pro-atherogenic in mice, we generated CCR5-deficient mice and crossed them with atherosclerosis-prone apoE-deficient mice. Although CCR5-deficient mice exhibit defects in induced macrophage trafficking, mean atherosclerotic lesion area did not differ significantly between apoE-deficient mice and apoE/CCR5-deficient mice after 16 weeks on a diet of normal chow. Ribonuclease protection assays (RPA) on RNA isolated from plaques from both apoE-deficient and apoE/CCR5-deficient animals showed strong signals for the macrophage marker F4/80 but no evidence for expression of prominent markers of T and B lymphocytes. These results indicate that the early stages of plaque formation in this model of lipid-mediated atherogenesis do not depend on CCR5.     

Hyperlipidemia and atherosclerotic lesion development in LDL receptor-deficient mice fed defined semipurified diets with and without cholate.

Past studies of atherosclerosis in mice have used chow-based diets supplemented with cholesterol, lipid, and sodium cholate to overcome species resistance to lesion formation. Similar diets have been routinely used in studies with LDL receptor-deficient (LDLR(-/-)) mice. The nonphysiological nature and potential toxicity of cholate-containing diets have led to speculation that atherogenesis in these mice may not accurately reflect the human disease process. We have designed a semipurified AIN-76A-based diet that can be fed in powdered, pelleted, or liquid form and manipulated for the precise evaluation of diet-genetic interactions in murine atherosclerosis. LDLR(-/-) mice were randomly assigned among 4 diets (n=6/diet) as follows: 1, control, 10% kcal lipid; 2, high fat (40% kcal), moderate cholesterol (0.5% by weight); 3, high fat, high cholesterol (1.25% by weight); and 4, high fat, high cholesterol, and 0.5% (wt/wt) sodium cholate. Fasting serum cholesterol was increased in all cholesterol-supplemented mice compared with controls after 6 or 12 weeks of feeding (P<0.01). The total area of oil red O-stained atherosclerotic lesions was determined from digitally scanned photographs. In contrast to the control group, all mice in cholesterol-supplemented dietary groups 2 to 4 had lesions involving 7.01% to 12.79% area of the thoracic and abdominal aorta at 12 weeks (P<0.002, for each group versus control). The distribution pattern of atherosclerotic lesions was highly reproducible and comparable. The histological features of lesions in mice fed cholate-free or cholate-containing diets were similar. This study shows that sodium cholate is not necessary for the formation of atherosclerosis in LDLR(-/-) mice and that precisely defined semipurified diets are a valuable tool for the examination of diet-gene interactions.     

Exercise reduces preexisting atherosclerotic lesions in LDL receptor knock out mice

Exercise is recommended both as a prophylactic and also as a therapeutic approach for patients with established coronary artery disease. In this study, we investigated the effect of a normal chow diet, with or without exercise in LDL r-/- mice with preexisting atherosclerotic lesions. A total of 28 LDL r-/- mice (LDL receptor knock out mice, 4-6 weeks old) were fed a high fat, high cholesterol diet (inductive phase). At the end of the 3 months, eight mice were sacrificed, and plasma autoantibodies to oxidatively modified proteins, cholesterol levels, and surface area of the lesions in the aorta were determined. The remaining mice were divided into two groups, and placed on a normal chow diet alone, or normal chow and exercise for three more months (regressive phase). Plasma autoantibodies to oxidatively modified proteins and cholesterol were measured along with the lesion size. Compared to the group of animals at the end of the inductive phase, both the groups of animals in the regressive phase had very low levels of plasma cholesterol and autoantibodies, and almost a 50% reduction in the aortic lesion area. The group that was exercised had the lowest levels of autoantibodies and aortic lesions as compared to the group without the exercise. However, the plasma cholesterol levels were comparable in both groups. This study demonstrates that reduction of preexisting atherosclerotic lesions is accelerated dramatically by exercise in LDL r-/- mice.     

Cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male LDL receptor knockout mice

ObjectiveIt is generally assumed that hepatic inflammation in obesity is linked to the pathogenesis of insulin resistance. Several recent studies have shed doubt on this view, which questions the causality of this association. This study focuses on Kupffer cell-mediated hepatic inflammation as a possible driver of insulin resistance in the absence and presence of obesity.MethodsWe used male mice deficient for the low-density lipoprotein receptor (Ldlr^−/−) and susceptible to cholesterol-induced hepatic inflammation. Whole body and hepatic insulin resistance was measured in mice fed 4 diets for 2 and 15 weeks, i.e., chow, high-fat (HF), HF-cholesterol (HFC; 0.2% cholesterol) and HF without cholesterol (HFnC). Biochemical parameters in plasma and liver were measured and inflammation was determined using immunohistochemistry and RT-PCR.ResultsAt 2 weeks, we did not find significant metabolic effects in either diet group, except for the mice fed a HFC diet which showed pronounced hepatic inflammation (p < 0.05) but normal insulin sensitivity. At 15 weeks, a significant increase in insulin levels, HOMA-IR, and hepatic insulin resistance was observed in mice fed a HFC, HFnC, and HF diet compared to chow-fed mice (p < 0.05). Regardless of the level of hepatic inflammation (HFC > HF, HFnC; p < 0.05) insulin resistance in mice fed HFC was no worse compared to mice on a HFnC and HF diet.ConclusionThese data show that cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male Ldlr^−/− mice. This study suggests that Kupffer cell-driven hepatic inflammation is a consequence, not a cause, of metabolic dysfunction in obesity.