Distinct Influence of Hypercaloric Diets Predominant with Fat or Fat and Sucrose on Adipose Tissue and Liver Inflammation in Mice.

Overfeeding of a hypercaloric diet leads to obesity, diabetes, chronic inflammation, and fatty liver disease. Although limiting fat or carbohydrate intake is the cornerstone for obesity management, whether lowering fat or reducing carbohydrate intake is more effective for health management remains controversial. This study used murine models to determine how dietary fat and carbohydrates may influence metabolic disease manifestation. Age-matched C57BL/6J mice were fed 2 hypercaloric diets with similar caloric content, one with very high fat and low carbohydrate content (VHF) and the other with moderately high fat levels with high sucrose content (HFHS) for 12 weeks. Both groups gained more weight and displayed hypercholesterolemia, hyperglycemia, hyperinsulinemia, and liver steatosis compared to mice fed a normal low-fat (LF) diet. Interestingly, the VHF-fed mice showed a more robust adipose tissue inflammation compared to HFHS-fed mice, whereas HFHS-fed mice showed liver fibrosis and inflammation that was not observed in VHF-fed mice. Taken together, these results indicate macronutrient-specific tissue inflammation with excess dietary fat provoking adipose tissue inflammation, whereas moderately high dietary fat with extra sucrose is necessary and sufficient for hepatosteatosis advancement to steatohepatitis. Hence, liver and adipose tissues respond to dietary fat and sucrose in opposite manners, yet both macronutrients are contributing factors to metabolic diseases.

Deficiency of LRP1 in Mature Adipocytes Promotes Diet-Induced Inflammation and Atherosclerosis-Brief Report.

OBJECTIVE: Mice with adipocyte-specific inactivation of low-density lipoprotein receptor-related protein-1 (LRP1) are resistant to diet-induced obesity and hyperglycemia because of compensatory thermogenic response by muscle. However, the physiological function of LRP1 in mature adipocytes and its role in cardiovascular disease modulation are unknown. This study compared perivascular adipose tissues (PVAT) from wild-type (adLrp1+/+) and adipocyte-specific LRP1 knockout (adLrp1-/-) mice in modulation of atherosclerosis progression. APPROACH AND RESULTS: Analysis of adipose tissues from adLrp1+/+ and adLrp1-/- mice after Western diet feeding for 16 weeks revealed that, in comparison to adLrp1+/+ mice, the adipocytes in adLrp1-/- mice were smaller, but their adipose tissues were more inflamed with increased monocyte-macrophage infiltration and inflammatory gene expression. The transplantation of PVAT from chow-fed adLrp1+/+ and adLrp1-/- mice into the area surrounding the carotid arteries of Ldlr-/- mice before feeding the Western diet revealed a contributory role of PVAT toward hypercholesterolemia-induced atherosclerosis. Importantly, recipients of adLrp1-/- PVAT displayed a 3-fold increase in atherosclerosis compared with adLrp1+/+ PVAT recipients. The increased atherosclerosis invoked by LRP1-deficient PVAT was associated with elevated monocyte-macrophage infiltration and inflammatory cytokine expression in the transplanted fat. CONCLUSIONS: PVAT provide outside-in signals through the adventitia to modulate atherosclerotic lesion progression in response to hypercholesterolemia. Moreover, adipocytes with LRP1 deficiency are dysfunctional and more inflamed. This latter observation adds the adipose tissue to the list of anatomic sites where LRP1 expression is important to protect against diet-induced atherosclerosis.

Bilirubin Prevents Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor-Deficient Mice by Inhibiting Endothelial VCAM-1 and ICAM-1 Signaling.

BACKGROUND: Numerous epidemiological studies support an inverse association between serum bilirubin levels and the incidence of cardiovascular disease; however, the mechanism(s) by which bilirubin may protect against atherosclerosis is undefined. The goals of the present investigations were to assess the ability of bilirubin to prevent atherosclerotic plaque formation in low-density lipoprotein receptor-deficient (Ldlr-/- ) mice and elucidate the molecular processes underlying this effect. METHODS AND RESULTS: Bilirubin, at physiological concentrations (≤20 µmol/L), dose-dependently inhibits THP-1 monocyte migration across tumor necrosis factor α-activated human umbilical vein endothelial cell monolayers without altering leukocyte binding or cytokine production. A potent antioxidant, bilirubin effectively blocks the generation of cellular reactive oxygen species induced by the cross-linking of endothelial vascular cell adhesion molecule 1 (VCAM-1) or intercellular adhesion molecule 1 (ICAM-1). These findings were validated by treating cells with blocking antibodies or with specific inhibitors of VCAM-1 and ICAM-1 signaling. When administered to Ldlr-/- mice on a Western diet, bilirubin (30 mg/kg intraperitoneally) prevents atherosclerotic plaque formation, but does not alter circulating cholesterol or chemokine levels. Aortic roots from bilirubin-treated animals exhibit reduced lipid and collagen deposition, decreased infiltration of monocytes and lymphocytes, fewer smooth muscle cells, and diminished levels of chlorotyrosine and nitrotyrosine, without changes in VCAM-1 or ICAM-1 expression. CONCLUSIONS: Bilirubin suppresses atherosclerotic plaque formation in Ldlr-/- mice by disrupting endothelial VCAM-1- and ICAM-1-mediated leukocyte migration through the scavenging of reactive oxygen species signaling intermediaries. These findings suggest a potential mechanism for the apparent cardioprotective effects of bilirubin.

Antiphospholipid antibodies attenuate endothelial repair and promote neointima formation in mice.

BACKGROUND: Antiphospholipid syndrome patients have antiphospholipid antibodies (aPLs) that promote thrombosis, and they have increased cardiovascular disease risk. Although the basis for the thrombosis has been well delineated, it is not known why antiphospholipid syndrome patients also have an increased prevalence of nonthrombotic vascular occlusion. The aims of this work were to determine if aPLs directly promote medial hypertrophy or neointima formation in mice and to identify the underlying mechanisms. METHODS AND RESULTS: Medial hypertrophy and neointima formation invoked by carotid artery endothelial denudation were evaluated in mice administered normal human IgG or aPLs. While aPLs had no effect on medial hypertrophy, they caused exaggerated neointima development. This was related to an aPL-induced impairment in reendothelialization post denudation, and scratch assays in cell culture revealed that there are direct effects of aPLs on endothelium that retard cell migration. Further experiments showed that aPL antagonism of endothelial migration and repair is mediated by antibody recognition of ß2-glycoprotein I, apolipoprotein E receptor 2, and a decline in bioavailable NO. Consistent with these mechanisms, the adverse impacts of aPLs on reendothelialization and neointima formation were fully prevented by the NO donor molsidomine. CONCLUSIONS: APLs blunt endothelial repair, and there is related aPL-induced exaggeration in neointima formation after endothelial injury in mice. The initiating process entails NO deficiency mediated by ß2-glycoprotein I recognition by aPLs and apolipoprotein E receptor 2. The modulation of endothelial apolipoprotein E receptor 2 function or NO bioavailability may represent new interventions to prevent the nonthrombotic vascular occlusion and resulting cardiovascular disorders that afflict antiphospholipid syndrome patients.

Genetic variants of ApoE and ApoER2 differentially modulate endothelial function.

It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE-ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte-endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2(-/-) mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2(-/-) mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function.

Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1.

OBJECTIVE: Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. APPROACH AND RESULTS: We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet-fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. CONCLUSIONS: These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1-dependent mechanisms.

Apolipoprotein E receptor-2 deficiency enhances macrophage susceptibility to lipid accumulation and cell death to augment atherosclerotic plaque progression and necrosis.

Genome-wide association studies have linked LRP8 polymorphisms to premature coronary artery disease and myocardial infarction in humans. However, the mechanisms by which dysfunctions of apolipoprotein E receptor-2 (apoER2), the protein encoded by LRP8 gene, influence atherosclerosis have not been elucidated completely. The current study focused on the role of apoER2 in macrophages, a cell type that plays an important role in atherosclerosis. Results showed that apoER2-deficient mouse macrophages accumulated more lipids and were more susceptible to oxidized LDL (oxLDL)-induced death compared to control cells. Consistent with these findings, apoER2 deficient macrophages also displayed defective serum-induced Akt activation and higher levels of the pro-apoptotic protein phosphorylated p53. Furthermore, the expression and activation of peroxisome proliferator-activated receptor γ (PPARγ) were increased in apoER2-deficient macrophages. Deficiency of apoER2 in hypercholesterolemic LDL receptor-null mice (Lrp8(-/-)Ldlr(-/-) mice) also resulted in accelerated atherosclerosis with more complex lesions and extensive lesion necrosis compared to Lrp8(+/+)Ldlr(-/-) mice. The atherosclerotic plaques of Lrp8(-/-)Ldlr(-/-) mice displayed significantly higher levels of p53-positive macrophages, indicating that the apoER2-deficient macrophages contribute to the accelerated atherosclerotic lesion necrosis observed in these animals. Taken together, this study indicates that apoER2 in macrophages limits PPARγ expression and protects against oxLDL-induced cell death. Thus, abnormal apoER2 functions in macrophages may at least in part contribute to the premature coronary artery disease and myocardial infarction in humans with LRP8 polymorphisms. Moreover, the elevated PPARγ expression in apoER2-deficient macrophages suggests that LRP8 polymorphism may be a genetic modifier of cardiovascular risk with PPARγ therapy.

Group 1B phospholipase A2 inactivation suppresses atherosclerosis and metabolic diseases in LDL receptor-deficient mice.

OBJECTIVE: Previous studies have shown that inactivation of the group 1B phospholipase A2 (Pla2g1b) suppresses diet-induced obesity, hyperglycemia, insulin resistance, and hyperlipidemia in C57BL/6 mice. A possible influence of Pla2g1b inactivation on atherosclerosis has not been addressed previously. The current study utilized LDL receptor-deficient (Ldlr(-/-)) mice with plasma lipid levels and distribution similar to hyperlipidemic human subjects as a preclinical animal model to test the effectiveness of Pla2g1b inactivation on atherosclerosis. METHODS AND RESULTS: The Pla2g1b(+/+)Ldlr(-/-) and Pla2g1b(-/-)Ldlr(-/-) mice were fed a low fat chow diet or a hypercaloric diet with 58.5 kcal% fat and 25 kcal% sucrose for 10 weeks. Minimal differences were observed between Pla2g1b(+/+)Ldlr(-/-) and Pla2g1b(-/-)Ldlr(-/-) mice when the animals were maintained on the low fat chow diet. However, when the animals were maintained on the hypercaloric diet, the Pla2g1(+/+)Ldlr(-/-) mice showed the expected body weight gain but the Pla2g1b(-/-)Ldlr(-/-) mice were resistant to diet-induced body weight gain. The Pla2g1b(-/-)Ldlr(-/-) mice also displayed lower fasting glucose, insulin, and plasma lipid levels compared to the Pla2g1b(+/+)Ldlr(-/-) mice, which displayed robust hyperglycemia, hyperinsulinemia, and hyperlipidemia in response to the hypercaloric diet. Importantly, atherosclerotic lesions in the aortic roots were also reduced 7-fold in the Pla2g1b(-/-)Ldlr(-/-) mice. CONCLUSION: The effectiveness of Pla2g1b inactivation to suppress diet-induced body weight gain and reduce diabetes and atherosclerosis in LDL receptor-deficient mice suggests that pharmacological inhibition of Pla2g1b may be a viable strategy to decrease diet-induced obesity and the risk of diabetes and atherosclerosis in humans.

Apolipoprotein E2 accentuates postprandial inflammation and diet-induced obesity to promote hyperinsulinemia in mice.

Genetic studies have revealed the association between the ε2 allele of the apolipoprotein E (apoE) gene and greater risk of metabolic diseases. This study compared C57BL/6 mice in which the endogenous mouse gene has been replaced by the human APOE2 or APOE3 gene (APOE2 and APOE3 mice) to identify the mechanism underlying the relationship between ε2 and obesity and diabetes. In comparison with APOE3 mice, the APOE2 mice had elevated fasting plasma lipid and insulin levels and displayed prolonged postprandial hyperlipidemia accompanied by increased granulocyte number and inflammation 2 h after being fed a lipid-rich meal. In comparison with APOE3 mice, the APOE2 mice also showed increased adiposity when maintained on a Western-type, high-fat, high-cholesterol diet. Adipose tissue dysfunction with increased macrophage infiltration, abundant crown-like structures, and inflammation were also observed in adipose tissues of APOE2 mice. The severe adipocyte dysfunction and tissue inflammation corresponded with the robust hyperinsulinemia observed in APOE2 mice after being fed the Western-type diet. Taken together, these data showed that impaired plasma clearance of apoE2-containing, triglyceride-rich lipoproteins promotes lipid redistribution to neutrophils and adipocytes to accentuate inflammation and adiposity, thereby accelerating the development of hyperinsulinemia that will ultimately lead to advanced metabolic diseases.

Myeloid-specific Krüppel-like factor 2 inactivation increases macrophage and neutrophil adhesion and promotes atherosclerosis.

RATIONALE: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. METHODS AND RESULTS: Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2(-/-)) mice were similar to myeKlf2(+/+) macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2(+/+) macrophages, myeKlf2(-/-) macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2(-/-) mice also adhered more robustly to endothelial cells, and fewer myeKlf2(-/-) neutrophils survived in culture over a 24-hour period in comparison with myeKlf2(+/+) neutrophils. When myeKlf2(-/-) mice were mated to Ldlr(-/-) mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2(-/-)Ldlr(-/-) mice compared with myeKlf2(+/+)Ldlr(-/-) littermates. The increased atherosclerosis in myeKlf2(-/-)Ldlr(-/-) mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2(+/+)Ldlr(-/-) mice. CONCLUSIONS: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.

Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice.

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.

Participation of ATP7A in macrophage mediated oxidation of LDL.

ATP7A primarily functions to egress copper from cells, thereby supplying this cofactor to secreted copper-accepting enzymes. This ATPase has attracted significant attention since the discovery of its mutation leading to human Menkes disease and the demonstration of its distribution in various tissues. Recently, we reported that ATP7A is expressed in the human vasculature. In the present study, we investigated the cellular expression of ATP7A in atherosclerotic lesions of LDL receptor (-/-) mice. Subsequently, we examined the role of ATP7A in regulating the oxidation of LDL in a macrophage cell model. We observed that ATP7A is expressed in atherosclerotic murine aorta and colocalizes with macrophages. To investigate the function of ATP7A, we downregulated ATP7A expression in THP-1 derived macrophages using small interfering RNA (siRNA). ATP7A downregulation attenuated cell-mediated oxidation of LDL. Moreover, downregulation of ATP7A resulted in decreased expression and enzymatic activity of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), a key intracellular enzyme involved in cell-mediated LDL oxidation. In addition, cPLA(2)alpha promoter activity was decreased after downregulation of ATP7A, suggesting that ATP7A transcriptionally regulates cPLA(2)alpha expression. Finally, cPLA(2)alpha overexpression increased LDL oxidation, which was blocked by coadministration of ATP7A siRNA oligonucleotides. These findings suggest a novel mechanism linking ATP7A to cPLA(2)alpha and LDL oxidation, suggesting that this copper transporter could play a previously unrecognized role in the pathogenesis of atherosclerosis.