Renee C. LeBoeuf

Diet-induced hypercholesterolemia enhances brain Aβ accumulation in transgenic mice

Epidemiological data show correlations between hypercholesterolemia and Alzheimers disease (AD). We test the hypothesis that hypercholesterolemia modulates A β deposition in mice overexpressing the human APP695 Swedish mutation (K670N and M671L) (TgAPPsw). Feeding mice a high fat/high cholesterol (HFHC) diet for 7–10 months increased total cholesterol levels by 4-fold. The extent of A β immunostained plaque-like deposits were significantly higher for mice fed the HFHC diet as compared with mice fed rodent chow. Extent of deposits correlated inversely with plasma levels of HDL and directly to apolipoprotein E. Overall, plasma lipoproteins may be an important factor in induction of AD-like plaques in mice. The lowering of plasma lipids may be therapeutic for AD patients.

INCREASED ATHEROSCLEROSIS IN STREPTOZOTOCIN-INDUCED DIABETIC MICE

Premature and extensive atheroscleroses involving renal, peripheral, and cardiovascular sites remain major complications of diabetes mellitus. Controversy exists as to the contribution of hyperglycemia versus elevated local or systemic concentrations of insulin to atherosclerosis risk. In this report, we developed the first murine model susceptible to both atherosclerosis and diabetes to determine which diabetogenic factors contribute to vascular disease. C57BL/6 and BALB/c mice were treated with multiple low-dose streptozotocin (STZ) or control citrate buffer and fed rodent chow or an atherogenic-promoting (Ath) diet for 12-20 wk. STZ treatment resulted in sustained hyperglycemia (250-420 mg/dl) and a modest reduction in plasma insulin levels for both strains regardless of diet. Citrate-treated C57BL/6 mice fed the Ath diet showed extensive oil red O-staining fatty streak aortic sinus lesions (20,537+/-2,957 micron2), the size of which did not differ for Ath-fed mice treated with STZ (16,836+/-2,136 micron2). In contrast, hyperglycemic BALB/c mice fed the Ath diet showed a 17-fold increase in atherosclerotic lesion area (7,922+/-2,096 micron2) as compared with citrate-treated mice fed the Ath diet (467+/-318 micron2). Correlations between lesion size and plasma glucose levels were significant for BALB/c (r = 0.741, P < 0.009), but not C57BL/6 (r = 0.314, P<0.3) mice. Lesion size correlated significantly with plasma cholesterol for C57BL/6 (r = 0.612, P<0.03) but not BALB/c (r = 0.630, P<0.1) mice. Immunohistochemistry showed that aortic sinus lesions from both strains contained macrophages, but smooth muscle cells were clearly present in lesions of BALB/c mice. In summary, we present the first small animal model showing accelerated atherosclerosis in response to hyperglycemia. Fatty streaks resembled those of human type II lesions in that both macrophages and smooth muscle cells were evident. In addition, our results support the concept that hyperglycemia as opposed to hyperinsulinemia contributes heavily to risk of atherosclerosis.

Impaired Superoxide Production Due to a Deficiency in Phagocyte NADPH Oxidase Fails to Inhibit Atherosclerosis in Mice

Superoxide, the reduced form of molecular oxygen, has been implicated in the genesis of vascular disease. One potential mechanism involves oxidation of low density lipoprotein into an atherogenic particle. A second involves reaction with nitric oxide to generate peroxynitrite, a highly oxidizing intermediate. A third involves regulation of signal transduction in artery wall cells. One well-characterized pathway for superoxide production resides in macrophages, the cellular hallmark of the early atherosclerotic lesion. Macrophages contain a membrane-bound NADPH oxidase that reduces oxygen to superoxide. In the current studies, we used mice that are deficient in the gp91-phox subunit of the NADPH oxidase-a model of chronic granulomatous disease (CGD)-to explore the role of superoxide in atherosclerotic vascular disease. Wild-type and CGD mice on the C57BL/6 background received a high-fat diet for 20 weeks to induce hypercholesterolemia. At the end of this period, the 2 strains of mice had comparable plasma lipid levels, and their atherosclerotic lesions were similar in size. We also crossed CGD mice with apolipoprotein E-deficient (apoE-/-) mice to generate spontaneously hypercholesterolemic animals that lacked functional NADPH oxidase. After 24 weeks, the CGD-apoE-/- animals had lower plasma cholesterol and triglyceride levels than did the apoE-/- animals, but there was no difference in the extent of atherosclerotic plaque. Our findings suggest that superoxide generated by the NADPH oxidase of phagocytes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia.

Obesity and diabetes in TNF-alpha receptor- deficient mice.

TNF-alpha may play a role in mediating insulin resistance associated with obesity. This concept is based on studies of obese rodents and humans, and cell culture models. TNF elicits cellular responses via two receptors called p55 and p75. Our purpose was to test the involvement of TNF in glucose homeostasis using mice lacking one or both TNF receptors. C57BL/6 mice lacking p55 (p55(-)/-), p75, (p75(-)/-), or both receptors (p55(-)/-p75(-)/-) were fed a high-fat diet to induce obesity. Marked fasting hyperinsulinemia was seen for p55(-)/-p75(-)/- males between 12 and 16 wk of feeding the high-fat diet. Insulin levels were four times greater than wild-type mice. In contrast, p55(-)/- and p75(-)/- mice exhibited insulin levels that were similar or reduced, respectively, as compared with wild-type mice. In addition, high-fat diet-fed p75(-)/- mice had the lowest body weights and leptin levels, and improved insulin sensitivity. Obese (db/db) mice, which are not responsive to leptin, were used to study the role of p55 in severe obesity. Male p55(-)/-db/db mice exhibited threefold higher insulin levels and twofold lower glucose levels at 20 wk of age than control db/db expressing p55. All db/db mice remained severely insulin resistant based on fasting plasma glucose and insulin levels, and glucose and insulin tolerance tests. Our data do not support the concept that TNF, acting via its receptors, is a major contributor to obesity-associated insulin resistance. In fact, data suggest that the two TNF receptors work in concert to protect against diabetes.

C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis

Non-insulin-dependent diabetes mellitus (NIDDM) is a major risk factor for the development of atherosclerosis in humans. The development of an animal model that displays accelerated atherosclerosis associated with NIDDM will aid in elucidating the mechanisms that associate these disorders. C57BL/6 mice may provide such a model system. This strain becomes obese, hyperglycemic and insulin resistant when fed a high fat diet (diabetogenic diet) and is susceptible to atherosclerotic lesion development when fed a separate high fat diet containing cholesterol and bile acids (atherogenic diet). This report tests whether a diet commonly used to induce atherosclerosis also provokes a diabetic phenotype and whether a diet used to induce diabetes provokes the development of aortic fatty streak lesions. Mice of strains C57BL/6, C3H/He, BALB/c and seven recombinant inbred (RI) strains were fed an atherogenic diet for 14 weeks and glucose parameters were measured. No correlation was observed between atherosclerosis susceptibility and fasting insulin or glucose levels, or glucose clearance following short-term insulin or glucose treatment. Analysis of the RI strains suggested that multiple genes control these glucose metabolic parameters. Feeding the diabetogenic diet for 14 weeks to C57BL/6 mice induced obesity and diabetes and 2-fold increases in plasma lipoprotein concentrations. Also, small aortic sinus lipid deposits were observed in 40% of the mice. Thus, analysis of the diabetogenic diet fed C57BL/6 mouse may provide an important tool for further studies of diabetes accelerated vascular disease.

Loss of Lymphotoxin-α but Not Tumor Necrosis Factor-α Reduces Atherosclerosis in Mice

Inflammatory processes are involved with all phases of atherosclerotic lesion growth. Tumor necrosis factor-α (TNFα) is an inflammatory cytokine that is thought to contribute to lesion development. Lymphotoxin-α (LTα) is also a proinflammatory cytokine with homology to TNFα. However, its presence or function in lesion development has not been investigated. To study the role of these molecules in atherosclerosis, the expression of these cytokines in atherosclerotic lesions was examined. The presence of both cytokines was observed within aortic sinus fatty streak lesions. To determine the function of these molecules in regulating lesion growth, mice deficient for TNFα or LTα were examined for induction of atherosclerosis. Surprisingly, loss of TNFα did not alter lesion development compared with wild-type mice. This brings doubt to the generally held concept that TNFα is a “proatherogenic cytokine.” However, LTα deficiency resulted in a 62% reduction in lesion size. This demonstrates an unexpected role for LTα in promoting lesion growth. The presence of LTα was observed in aortic sinus lesions suggesting a direct role of LTα in modulating lesion growth. To determine which receptor mediated these responses, diet-induced atherosclerosis in mice deficient for each of the TNF receptors, termed p55 and p75, was examined. Results demonstrated that loss of p55 resulted in increased lesion development, but loss of p75 did not alter lesion size. The disparity in results between ligand- and receptor-deficient mice suggests there are undefined members of the TNF ligand and receptor signaling pathway involved with regulating atherogenesis.

Expression of Human Myeloperoxidase by Macrophages Promotes Atherosclerosis in Mice

Background—Myeloperoxidase (MPO) colocalizes with macrophages in the human artery wall, and its characteristic oxidation products have been detected in atherosclerotic lesions. Thus, oxidants produced by the enzyme might promote atherosclerosis. However, macrophages in mouse atherosclerotic tissue do not express MPO. Therefore, mice are an inappropriate model for testing the role of MPO in vascular disease. To overcome this problem, we generated and studied transgenic (Tg) mice that contained the human MPO gene. Methods and Results—We produced human MPO-Tg mice with use of a Visna virus promoter. To confine MPO expression to macrophages, we lethally irradiated LDL receptor–deficient mice and repopulated their bone marrow with cells from wild-type mice or MPO-Tg mice. Despite having similarly high levels of cholesterol after maintenance on a high-fat, high-cholesterol diet, the MPO-Tg animals developed a 2-fold greater atherosclerotic area in the aorta than did mice transplanted with wild-type bone marrow (P=0.00003). Conclusions—Our observations indicate that expression of human MPO in macrophages promotes atherosclerosis in hypercholesterolemic mice, raising the possibility that the enzyme might be a potential therapeutic target for preventing cardiovascular disease in humans.

β-Cell-Specific Overexpression of Glutathione Peroxidase Preserves Intranuclear MafA and Reverses Diabetes in db/db Mice

Chronic hyperglycemia causes oxidative stress, which contributes to damage in various tissues and cells, including pancreatic beta-cells. The expression levels of antioxidant enzymes in the islet are low compared with other tissues, rendering the beta-cell more susceptible to damage caused by hyperglycemia. The aim of this study was to investigate whether increasing levels of endogenous glutathione peroxidase-1 (GPx-1), specifically in beta-cells, can protect them against the adverse effects of chronic hyperglycemia and assess mechanisms that may be involved. C57BLKS/J mice overexpressing the antioxidant enzyme GPx-1 only in pancreatic beta-cells were generated. The biological effectiveness of the overexpressed GPx-1 transgene was documented when beta-cells of transgenic mice were protected from streptozotocin. The transgene was then introgressed into the beta-cells of db/db mice. Without use of hypoglycemic agents, hyperglycemia in db/db-GPx(+) mice was initially ameliorated compared with db/db-GPx(-) animals and then substantially reversed by 20 wk of age. beta-Cell volume and insulin granulation and immunostaining were greater in db/db-GPx(+) animals compared with db/db-GPx(-) animals. Importantly, the loss of intranuclear musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) that was observed in nontransgenic db/db mice was prevented by GPx-1 overexpression, making this a likely mechanism for the improved glycemic control. These studies demonstrate that enhancement of intrinsic antioxidant defenses of the beta-cell protects it against deterioration during hyperglycemia.

Dietary Antioxidants Inhibit Development of Fatty Streak Lesions in the LDL Receptor–Deficient Mouse

Oxidized low density lipoprotein (LDL) promotes atherogenesis. Although pharmacological antioxidants such as probucol inhibit both LDL oxidation and atherosclerosis in hyperlipidemic animals, the effects of natural antioxidants such as vitamin E are inconclusive. To further determine the effects of supplemental dietary antioxidants in vivo, we evaluated whether combined dietary antioxidants (0.1% vitamin E, 0.5% beta-carotene, and 0.05% vitamin C) inhibit LDL oxidation and fatty streak lesion development in homozygous LDL receptor-null (LDLR-/-) mice fed a high-fat, high-cholesterol diet. An additional group of mice were fed black tea, which has been shown to inhibit LDL oxidation in vitro. After receiving a high-fat, high-cholesterol diet for 8 weeks, the combined antioxidant-supplemented (antioxidant) group (n=18), tea group (n=19), and control group (n=17) had equivalent plasma cholesterol levels. LDL oxidation, as measured by the lag phase of conjugated diene formation, was markedly inhibited in the antioxidant group compared with the tea or control groups [mean lag phases=143+/-7 (antioxidant), 100+/-5 (tea), and 84+/-4 (control) minutes; P<0.0001 antioxidant versus tea or control]. The cross-sectional surface area of fatty streak lesions in the aortic sinus was reduced by 60% in the antioxidant group compared with both the tea and control groups (P<0.0001 antioxidant versus tea or control). There was no difference in lesion area between tea and control groups. Although both LDL oxidation and atherosclerosis were significantly inhibited in the antioxidant group, no correlation between lag phase values and lesion size was observed among individual animals. Furthermore, black tea did not inhibit fatty streak development in LDLR-/- mice. These data suggest that combined natural dietary antioxidants inhibit both LDL oxidation and atherogenesis in animals with elevated LDL but that inhibition of LDL oxidation alone may not prevent the development of atherosclerosis.

Renal injury in apolipoprotein E-deficient mice.

Hyperlipidemia is thought to accelerate the progression of renal diseases, but the mechanisms by which hyperlipidemia exerts its deleterious effect is still poorly understood. The aim of this study was to describe the renal pathology in a hyperlipidemic mouse strain, the apolipoprotein E–deficient mice (apoE−/−). Renal specimens from a total of 34 mice were studied, including 19 apoE−/− females at the age of 36 weeks, 9 apoE−/− females at the age of 24 weeks, and 6 wild-type females (C57BL/6) as controls. Kidneys were evaluated by histologic examination, immunohistochemistry, and electron microscopy. Immunohistochemistry was used to detect MAC-2–expressing monocyte/macrophages, and the proliferation marker PCNA. Glomerular cell number, glomerular matrix area, and glomerular area were quantified by morphometry. Glomerular lesions in apoE−/− mice were characterized by macrophage accumulation, commonly with foam cell appearance, deposition of extracellular matrix, glomerular hyperplasia, and at times prominent mesangiolysis associated with capillary microaneurysms. Some cases demonstrated lipid deposits filling glomerular capillaries. Arterioles of the vascular pole demonstrated a “foamy” degeneration of smooth muscle cells. These lesions related to hyperlipidemia in this well-established mouse strain have not been previously described. Because this mouse strain is among the most widely studied for interventions aimed at altering hyperlipidemia and the progression of atherosclerosis, we believe that our observations may be of major importance for the accurate interpretation of interventional studies in this strain and offer a new opportunity to study mechanisms of hyperlipidemic renal injury.