BACKGROUND: Obesity, an inflammatory condition linked to cardiovascular disease, is associated with expansion of adipose tissue. Highly prevalent coplanar polychlorinated biphenyls (PCBs) such as 3,3´,4,4´-tetrachlorobiphenyl (PCB-77) accumulate in adipose tissue because of their lipophilicity and increase with obesity. However, the effects of PCBs on adipocytes, obesity, and obesity-associated cardiovascular disease are unknown. OBJECTIVES: In this study we examined in vitro and in vivo effects of PCB-77 on adipocyte differentiation, proinflammatory adipokines, adipocyte morphology, body weight, serum lipids, and atherosclerosis. METHODS: PCB-77 or 2,2´,4,4,5,5´-hexachlorobiphenyl (PCB-153) was incubated with 3T3-L1 adipocytes either during differentiation or in mature adipocytes. Concentration-dependent effects of PCB-77 were contrasted with those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For in vivo studies, we treated C57BL/6 wild-type (WT) or aryl hydrocarbon receptor (AhR) -/mice with vehicle or PCB-77 (49 mg/kg, by intraperitoneal injection) and examined body weight gain. In separate studies, we injected ApoE -/mice with vehicle or PCB-77 over a 6-week period and examined body weight, adipocyte size, serum lipids, and atherosclerosis. RESULTS: Low concentrations of PCB-77 or TCDD increased adipocyte differentiation, glycerol-3phosphate dehydrogenase activity, and expression of peroxisome proliferator-activated receptor γ, whereas higher concentrations inhibited adipocyte differentiation. Effects of PCB-77 were abolished by the AhR antagonist α-naphthoflavone. PCB-77 promoted the expression and release of various proinflammatory cytokines from 3T3-L1 adipocytes. Administration of PCB-77 increased body weight gain in WT but not AhR -/mice. ApoE -/mice injected with PCB-77 exhibited greater body weight, adipocyte hypertrophy, serum dyslipidemia, and augmented atherosclerosis. CONCLUSIONS: Our findings suggest that PCB-77 may contribute to the development of obesity and obesity-associated atherosclerosis. KEY WORDS: adipocyte differentiation, aryl hydrocarbon receptor, ectopic lipid deposition, obesity, polychlorinated biphenyl.

and mouse atherosclerotic lesions, although its effects on the development of the disease are undefined. We determined the role of IL-4 in the most commonly used murine models of atherosclerosis by defining the effects of exogenous delivery and genetic deficiency of this cytokine on both hypercholesterolemia and AngII-induced atherosclerosis in apolipoprotein E (apoE) ؊/؊ mice and different dietary stimuli in low-density lipoprotein (LDL) receptor ؊/؊ mice. Exogenous administration of IL-4 (1.1 ng g ؊1 day ؊1 i.p. for 30 days) into female apoE ؊/؊ mice had no effect on lesion size or composition in mice fed normal or saturated fat diets. Also, IL-4 deficiency had no significant effect on the size or composition of atherosclerotic lesions in two vascular areas of male and female apoE ؊/؊ mice fed either a normal or saturated fat diet. IL-4 deficiency was also studied in age-matched male mice infused with AngII (1000 ng kg ؊1 min ؊1 ) for 28 days. Whereas AngII infusion augmented atherosclerotic lesion formation, IL-4 deficiency did not influence atherosclerotic lesion size or composition. Finally, different dietary stimuli also had no effect on atherosclerotic lesion size in female LDL receptor ؊/؊ mice. These data demonstrate that IL-4 does not significantly influence the development of atherosclerotic lesions in apoE ؊/؊ mice of either gender or in female LDL receptor ؊/؊ mice, irrespective of the mode of induction of atherosclerosis. (Am J Pathol 2007,

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-4Ϫ/Ϫ or IL-4ϩ/ϩ mice were transplanted into lethally irradiated female low density lipoprotein (LDL) receptorϪ/Ϫ 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 (9.1Ϯ1.1% versus 2.8Ϯ0.8% of intimal area, PϽ0.001) and the thoracic aorta (1.2Ϯ0.2% versus 0.4Ϯ0.1%, PϽ0.002). Therefore, IL-4 deficiency reduced atherosclerotic lesion formation in a site-specific manner in female LDL receptorϪ/Ϫ mice fed a high-fat diet. (Arterioscler Thromb Vasc Biol. 2002;22:456-461.)

1 We have demonstrated previously that infusion of angiotensin II (AngII) into hyperlipidemic mice augments atherosclerosis and results in the formation of abdominal aortic aneurysms (AAA). The purpose of this study was to determine the role of aldosterone in these AngII-induced vascular pathologies. 2 Male apolipoprotein EÀ/À (apoE) mice were infused with either vehicle or aldosterone (50 or 200 ng kg À1 min À1 ). Arterial blood pressure was determined throughout the study and serum lipid concentrations and vascular pathology were quantified after 28 days of infusion.

This review will focus on the role of cytokines in the behavior of macrophages, a prominent cell type of atherosclerotic lesions. Once these macrophages have immigrated into the vessel wall, they propagate the development of atherosclerosis by modifying lipoproteins, accumulating intracellular lipids, remodeling the extracellular environment, and promoting local coagulation. The numerous cytokines that have been detected in atherosclerosis, combined with the expression of large numbers of cytokine receptors on macrophages, are consistent with this axis being an important contributor to lesion development. Given the vast literature on cytokine-macrophage interactions, this review will be selective, with an emphasis on the major cytokines that have been detected in atherosclerotic lesions and their effects on properties that are relevant to lesion formation and maturation. There will be an emphasis on the role of cytokines in regulating lipid metabolism by macrophages. We will provide an overview of the major findings in cell culture and then put these in the context of in vivo studies. -Daugherty, A., N. R. Webb, D. L. Rateri, and V. L. King. Cytokine regulation of macrophage functions in atherogenesis. J. Lipid Res. 2005. 46: 1812-1822.

Background-Vascular disease can manifest as stenotic plaques or ectatic aneurysms, although the mechanisms culminating in these divergent disease manifestations remain poorly understood. T-helper type 1 cytokines, including interferon-␥ and CXCL10, have been strongly implicated in atherosclerotic plaque development. Methods and Results-Here, we specifically examined their role in the formation of abdominal aortic aneurysms in the angiotensin II-induced murine model. Unexpectedly, we found increased suprarenal aortic diameters, abdominal aortic aneurysm incidence, and aneurysmal death in apolipoprotein E-and interferon-␥-deficient (Apoe Ϫ/Ϫ /Ifng Ϫ/Ϫ ) mice compared with Apoe Ϫ/Ϫ controls, although atherosclerotic luminal plaque formation was attenuated. The interferon-␥inducible T-cell chemoattractant CXCL10 was highly induced by angiotensin II infusion in Apoe Ϫ/Ϫ mice, but this induction was markedly attenuated in Apoe Ϫ/Ϫ /Ifng Ϫ/Ϫ mice. Apoe Ϫ/Ϫ /Cxcl10 Ϫ/Ϫ mice had decreased luminal plaque but also increased aortic size, worse morphological grades of aneurysms, and a higher incidence of death due to aortic rupture than Apoe Ϫ/Ϫ controls. Furthermore, abdominal aortic aneurysms in Apoe Ϫ/Ϫ /Cxcl10 Ϫ/Ϫ mice were enriched for non-T-helper type 1-related signals, including transforming growth factor-␤1. Treatment of Apoe Ϫ/Ϫ /Cxcl10 Ϫ/Ϫ mice with anti-transforming growth factor-␤ neutralizing antibody diminished angiotensin II-induced aortic dilation. Conclusions-The present study defines a novel pathway in which interferon-␥ and its effector, CXCL10, contribute to divergent pathways in abdominal aortic aneurysm versus plaque formation, inhibiting the former pathology but promoting the latter. Thus, efforts to develop antiinflammatory strategies for atherosclerosis must carefully consider potential effects on all manifestations of vascular disease. (Circulation. 2009;119:426-435.)

Feeding interleukin-4 (IL-4) deficient C57BL/6 LDL receptor (LDLr) ؊ / ؊ mice a modified diet to investigate the role of this cytokine in cholesterol metabolism led to an unexpected phenotype. IL-4 ؊ / ؊ → LDLr ؊ / ؊ mice had enlarged gallbladders and an increased mortality that was preceded by acute body weight loss. To determine if IL-4 deficiency accounted for these findings, C57BL/6 IL-4 ؉ / ؉ and IL-4 ؊ / ؊ mice were fed either a normal or modified diet. IL-4 deficiency did not alter bile composition or cause liver toxicity in mice fed a fat-enriched diet. Following 8 weeks of feeding a fat-enriched diet, no gallstones were detected in IL-4 ؉ / ؉ mice, and only 20% had cholesterol crystals. In contrast, IL-4 ؊ / ؊ mice had a 100% incidence of gallstones and cholesterol crystals. IL-4 ؊ / ؊ deficiency also increased serum concentrations of bilirubin following feeding a fat-enriched diet. Therefore, these studies revealed an unexpected finding that IL-4 deficiency predisposes to gallstone formation. -King, V. L., S. J. Szilvassy, and A. Daugherty. Interleukin-4 deficiency promotes gallstone formation. J. Lipid Res. 2002. 43: 768-771. Supplementary key words bone marrow transplant • LDL receptor • cholesterol

Oxidized LDL induces changes in several facets of the immune system, although the relationships between these facets and their contributions to atherogenesis have yet to be fully elucidated. A report in this issue of the JCI provides a novel demonstration of the adaptive immune system influencing the production of natural antibodies. The results demonstrate that injection of malondialdehyde-modified LDL promotes a Th2 response that in turn increases the titers of the natural antibody T15/EO6, which recognizes the oxidized phospholipid POVPC. Atherosclerotic lesion size in LDL receptor-deficient mice is reduced as a consequence of the increase in natural antibody titers, and IL-5 is identified as the link between the adaptive and natural immune systems (see related article beginning on page 427).

The epidemic of obesity sweeping developed nations is accompanied by an increase in atherosclerotic cardiovascular diseases. Dyslipidemia, diabetes, hypertension, and obesity are risk factors for cardiovascular disease. However, delineating the mechanism of obesity-accelerated atherosclerosis has been hampered by a paucity of animal models. Similar to humans, apolipoprotein E–deficient (apoE−/−) mice spontaneously develop atherosclerosis over their lifetime. To determine whether apoE−/− mice would develop obesity with accelerated atherosclerosis, we fed mice diets containing 10 (low fat (LF)) or 60 (high fat (HF)) kcal % from fat for 17 weeks. Mice fed the HF diet had a marked increase in body weight and atherosclerotic lesion formation compared to mice fed the LF diet. There were no significant differences between groups in serum total cholesterol, triglycerides, or leptin concentrations. Plasma concentrations of the acute-phase reactant serum amyloid A (SAA) are elevated in both obesity and cardiovascular disease. Accordingly, plasma SAA concentrations were increased fourfold (P < 0.01) in mice fed the HF diet. SAA was associated with both pro- and antiatherogenic lipoproteins in mice fed the HF diet compared to those fed the LF diet, in which SAA was primarily associated with the antiatherogenic lipoprotein high-density lipoprotein (HDL). Moreover, SAA was localized with apoB-containing lipoproteins and biglycan in the vascular wall. Taken together, these data suggest male apoE-deficient mice are a model of metabolic syndrome and that chronic low level inflammation associated with increased SAA concentrations may mediate atherosclerotic lesion formation.

C-reactive protein (CRP) are predictive of cardiac disease and are proposed to play causal roles in the development of atherosclerosis, in which the retention of lipoproteins by vascular wall proteoglycans is critical. The purpose of this study was to determine whether SAA and/or CRP alters vascular proteoglycan synthesis and lipoprotein retention in a pro-atherogenic manner. Vascular smooth muscle cells were stimulated with either SAA or CRP (1 to 100 mg/L) and proteoglycans were then isolated and characterized. SAA, but not CRP, increased proteoglycan sulfate incorporation by 50 to 100% in a dose-dependent manner (P < 0.0001), increased glycosaminoglycan chain length, and increased low-density lipoprotein (LDL) binding affinity (K d , 29 g/ml LDL versus 90 g/ml LDL for SAA versus control proteoglycans; P < 0.005). Furthermore, SAA up-regulated biglycan via the induction of endogenous transforming growth factor (TGF)-␤. To determine whether SAA stimulated proteoglycan synthesis in vivo, ApoE ؊/؊ mice were injected with an adenovirus expressing human SAA-1, a null virus, or saline. Mice that received adenovirus expressing SAA had increased TGF-␤ concentrations in plasma and increased aortic biglycan content compared with mice that received either null virus or saline. Thus, SAA alters vascular proteoglycans in a pro-atherogenic manner via the stimulation of TGF-␤ and may play a causal role in the development of atherosclerosis.

Glucosamine, commonly consumed for the treatment of osteoarthritis, is classified as a nutritional supplement; however, there are few data regarding its metabolic or vascular effects. Glucosamine is a component of the hexosamine pathway, which has been implicated in the development of insulin resistance. Anecdotal reports suggest that glucosamine consumption can increase circulating cholesterol concentrations. To investigate the metabolic and vascular effects of glucosamine supplementation, we studied male and female LDL receptor-deficient mice fed a Western diet (21% fat, 0.15% cholesterol). Three groups of 6-10 mice of each gender received either no supplement, 15 mg Á kg 21 Á d 21 glucosamine (equivalent to an average human dose), or 50 mg Á kg 21 Á d 21 glucosamine added to their drinking water for 5, 10, or 20 wk. Plasma cholesterol and triglyceride concentrations increased in all mice with the addition of the Western diet.