Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr^−/− mice reconstituted with Akt1^−/− fetal liver cells (Akt1^−/−→Ldlr^−/−) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr^−/−). In contrast, Akt2^−/−→Ldlr^−/− mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr^−/− mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2^−/−→Ldlr^−/− mice had smaller aortic lesions compared with WT→Ldlr^−/− and Akt1^−/−→Ldlr^−/− mice. Importantly, Akt2^−/−→Ldlr^−/− mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6C^hi and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2^−/− mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.     

Raised Soluble P-Selectin Moderately Accelerates Atherosclerotic Plaque Progression

Soluble P-selectin (sP-selectin), a biomarker of inflammatory related pathologies including cardiovascular and peripheral vascular diseases, also has pro-atherosclerotic effects including the ability to increase leukocyte recruitment and modulate thrombotic responses in vivo. The current study explores its role in progressing atherosclerotic plaque disease. Apoe ^−/− mice placed on a high fat diet (HFD) were given daily injections of recombinant dimeric murine P-selectin (22.5 µg/kg/day) for 8 or 16 weeks. Saline or sE-selectin injections were used as negative controls. In order to assess the role of sP-selectin on atherothrombosis an experimental plaque remodelling murine model, with sm22α-hDTR Apoe^−/− mice on a HFD in conjunction with delivery of diphtheria toxin to induce targeted vascular smooth muscle apoptosis, was used. These mice were similarly given daily injections of sP-selectin for 8 or 16 weeks. While plaque mass and aortic lipid content did not change with sP-selectin treatment in Apoe^−/− or SM22α-hDTR Apoe^−/− mice on HFD, increased plasma MCP-1 and a higher plaque CD45 content in Apoe ^−/− HFD mice was observed. As well, a significant shift towards a more unstable plaque phenotype in the SM22α-hDTR Apoe^−/− HFD mice, with increased macrophage accumulation and lower collagen content, leading to a lower plaque stability index, was observed. These results demonstrate that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.     

Reduced VLDL clearance in Apoe−/−Npc1−/− mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe^−/−Npc1^−/− mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe^−/−Npc1^−/− liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe^−/−Npc1^−/− liver was unexpected. However, several other LXR target genes also increased in Apoe^−/−Npc1^−/− liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe^−/− mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe^−/−Npc1^−/− mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.     

Amelioration of Hyperglycemia with a Sodium-Glucose Cotransporter 2 Inhibitor Prevents Macrophage-Driven Atherosclerosis through Macrophage Foam Cell Formation Suppression in Type 1 and Type 2 Diabetic Mice

Direct associations between hyperglycemia and atherosclerosis remain unclear. We investigated the association between the amelioration of glycemia by sodium-glucose cotransporter 2 inhibitors (SGLT2is) and macrophage-driven atherosclerosis in diabetic mice. We administered dapagliflozin or ipragliflozin (1.0 mg/kg/day) for 4-weeks to apolipoprotein E-null (Apoe ^−/−) mice, streptozotocin-induced diabetic Apoe ^−/− mice, and diabetic db/db mice. We then determined aortic atherosclerosis, oxidized low-density lipoprotein (LDL)-induced foam cell formation, and related gene expression in exudate peritoneal macrophages. Dapagliflozin substantially decreased glycated hemoglobin (HbA1c) and glucose tolerance without affecting body weight, blood pressure, plasma insulin, and lipids in diabetic Apoe ^−/− mice. Aortic atherosclerotic lesions, atheromatous plaque size, and macrophage infiltration in the aortic root increased in diabetic Apoe ^−/− mice; dapagliflozin attenuated these changes by 33%, 27%, and 20%, respectively. Atherosclerotic lesions or foam cell formation highly correlated with HbA1c. Dapagliflozin did not affect atherosclerosis or plasma parameters in non-diabetic Apoe ^−/− mice. In db/db mice, foam cell formation increased by 4-fold compared with C57/BL6 mice, whereas ipragliflozin decreased it by 31%. Foam cell formation exhibited a strong correlation with HbA1c. Gene expression of lectin-like ox-LDL receptor-1 and acyl-coenzyme A:cholesterol acyltransferase 1 was upregulated, whereas that of ATP-binding cassette transporter A1 was downregulated in the peritoneal macrophages of both types of diabetic mice. SGLT2i normalized these gene expressions. Our study is the first to demonstrate that SGLT2i exerts anti-atherogenic effects by pure glucose lowering independent of insulin action in diabetic mice through suppressing macrophage foam cell formation, suggesting that foam cell formation is highly sensitive to glycemia ex vivo.     

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3⁺ regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4⁺ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr^−/−Pd1^−/−) displayed striking increases in systemic CD4⁺ and CD8⁺ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3⁺ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr^−/−Pd1^−/− mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr^−/− mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.     

Time-restricted feeding prevents high-fat and high-cholesterol diet-induced obesity but fails to ameliorate atherosclerosis in apolipoprotein E-knockout mice

One of the leading risk factors for atherosclerosis is obesity, which is commonly caused by a nutrient-rich Western-style diet, sedentary behaviors, and shift work. Time-restricted (TR) feeding and intermittent fasting are both known to prevent overweight and adiposity, improve glucose tolerance, and decrease plasma cholesterol in high-fat diet-induced obese mice. Here we examined the overall effects of TR feeding of a Western diet (fat, 40.5 Kcal%; cholesterol, 0.21 g%) using 8-week-old Apoe^−/− mice. Mice were assigned into three groups: (1) an ad libitum (AL) group fed an AL Western diet, (2) a TR group with restricted access to a Western diet (15 h/day, 12:00 to 3:00 Zeitgeber time [ZT]); and (3) an Ex/TR group fed a TR Western diet and subjected to physical exercise at 12:00 ZT. Mice in the AL group gained body weight rapidly during the 14-week observation period. With TR feeding, excessive weight gain, liver adiposity, visceral fat, and brown adipose tissue volume were effectively suppressed. Although TR feeding failed to decrease Oil Red O-stained aortic plaques in Apoe^−/− mice, physical exercise significantly decreased them. Neither TR feeding with exercise nor that without exercise decreased the mean area under the curve of the plasma cholesterol level or the fasting plasma glucose. Collectively, TR feeding of a Western diet prevented the development of obesity but failed to ameliorate atherosclerosis in Apoe^−/− mice.     

Preventive Effect of Dipeptidyl Peptidase-4 Inhibitor on Atherosclerosis Is Mainly Attributable to Incretin's Actions in Nondiabetic and Diabetic Apolipoprotein E-Null Mice

Aim

Several recent reports have revealed that dipeptidyl peptidase (DPP)-4 inhibitors have suppressive effects on atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. It remains to be seen, however, whether this effect stems from increased levels of the two active incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).

Methods

Nontreated Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db diabetic mice were administered the DPP-4 inhibitor vildagliptin in drinking water and co-infused with either saline, the GLP-1 receptor blocker, exendin(9–39), the GIP receptor blocker, (Pro³)GIP, or both via osmotic minipumps for 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages were determined.

Results

Vildagliptin increased plasma GLP-1 and GIP levels without affecting food intake, body weight, blood pressure, or plasma lipid profile in any of the animals tested, though it reduced HbA1c in the diabetic mice. Diabetic Apoe ^−/− mice exhibited further-progressed atherosclerotic lesions and foam cell formation compared with nondiabetic counterparts. Nondiabetic and diabetic Apoe ^−/− mice showed a comparable response to vildagliptin, namely, remarkable suppression of atherosclerotic lesions with macrophage accumulation and foam cell formation in peritoneal macrophages. Exendin(9–39) or (Pro³)GIP partially attenuated the vildagliptin-induced suppression of atherosclerosis. The two blockers in combination abolished the anti-atherosclerotic effect of vildagliptin in nondiabetic mice but only partly attenuated it in diabetic mice. Vildagliptin suppressed macrophage foam cell formation in nondiabetic and diabetic mice, and this suppressive effect was abolished by infusions with exendin(9–39)+(Pro³)GIP. Incubation of DPP-4 or vildagliptin in vitro had no effect on macrophage foam cell formation.

Conclusions

Vildagliptin confers a substantial anti-atherosclerotic effect in both nondiabetic and diabetic mice, mainly via the action of the two incretins. However, the partial attenuation of atherosclerotic lesions by the dual incretin receptor antagonists in diabetic mice implies that vildagliptin confers a partial anti-atherogenic effect beyond that from the incretins.     

Inhibition of intestinal cholesterol absorption decreases atherosclerosis but not adipose tissue inflammation

Adipose tissue inflammation is associated with insulin resistance and increased cardiovascular disease risk in obesity. We previously showed that addition of cholesterol to a diet rich in saturated fat and refined carbohydrate significantly worsens dyslipidemia, insulin resistance, adipose tissue macrophage accumulation, systemic inflammation, and atherosclerosis in LDL receptor-deficient (Ldlr^−/−) mice. To test whether inhibition of intestinal cholesterol absorption would improve metabolic abnormalities and adipose tissue inflammation in obesity, we administered ezetimibe, a dietary and endogenous cholesterol absorption inhibitor, to Ldlr^−/− mice fed chow or high-fat, high-sucrose (HFHS) diets without or with 0.15% cholesterol (HFHS+C). Ezetimibe blunted weight gain and markedly reduced plasma lipids in the HFHS+C group. Ezetimibe had no effect on glucose homeostasis or visceral adipose tissue macrophage gene expression in the HFHS+C fed mice, although circulating inflammatory markers serum amyloid A (SSA) and serum amyloid P (SSP) levels decreased. Nevertheless, ezetimibe treatment led to a striking (>85%) reduction in atherosclerotic lesion area with reduced lesion lipid and macrophage content in the HFHS+C group. Thus, in the presence of dietary cholesterol, ezetimibe did not improve adipose tissue inflammation in obese Ldlr^−/− mice, but it led to a major reduction in atherosclerotic lesions associated with improved plasma lipids and lipoproteins.     

Increased levels of invariant natural killer T lymphocytes worsen metabolic abnormalities and atherosclerosis in obese mice

Obesity is a chronic inflammatory state characterized by infiltration of adipose tissue by immune cell populations, including T lymphocytes. Natural killer T (NKT) cells, a specialized lymphocyte subset recognizing lipid antigens, can be pro- or anti-inflammatory. Their role in adipose inflammation continues to be inconclusive and contradictory. In obesity, the infiltration of tissues by invariant NKT (iNKT) cells is decreased. We therefore hypothesized that an excess iNKT cell complement might improve metabolic abnormalities in obesity. Vα14 transgenic (Vα14tg) mice, with increased iNKT cell numbers, on a LDL receptor-deficient (Ldlr^−/−) background and control Ldlr^−/− mice were placed on an obesogenic diet for 16 weeks. Vα14tg.Ldlr^−/− mice gained 25% more weight and had increased adiposity than littermate controls. Transgenic mice also developed greater dyslipidemia, hyperinsulinemia, insulin resistance, and hepatic triglyceride accumulation. Increased macrophage Mac2 immunostaining and proinflammatory macrophage gene expression suggested worsened adipose inflammation. Concurrently, these mice had increased atherosclerotic lesion area and aortic inflammation. Thus, increasing the complement of iNKT cells surprisingly exacerbated the metabolic, inflammatory, and atherosclerotic features of obesity. These findings suggest that the reduction of iNKT cells normally observed in obesity may represent a physiological attempt to compensate for this inflammatory condition.     

The low density lipoprotein receptor modulates the effects of hypogonadism on diet-induced obesity and related metabolic perturbations

Here, we investigated how LDL receptor deficiency (Ldlr^−/−) modulates the effects of testosterone on obesity and related metabolic dysfunctions. Though sham-operated Ldlr^−/− mice fed Western-type diet for 12 weeks became obese and showed disturbed plasma glucose metabolism and plasma cholesterol and TG profiles, castrated mice were resistant to diet-induced obesity and had improved glucose metabolism and reduced plasma TG levels, despite a further deterioration in their plasma cholesterol profile. The effect of hypogonadism on diet-induced weight gain of Ldlr^−/− mice was independent of ApoE and Lrp1. Indirect calorimetry analysis indicated that hypogonadism in Ldlr^−/− mice was associated with increased metabolic rate. Indeed, mitochondrial cytochrome c and uncoupling protein 1 expression were elevated, primarily in white adipose tissue, confirming increased mitochondrial metabolic activity due to thermogenesis. Testosterone replacement in castrated Ldlr^−/− mice for a period of 8 weeks promoted diet-induced obesity, indicating a direct role of testosterone in the observed phenotype. Treatment of sham-operated Ldlr^−/− mice with the aromatase inhibitor exemestane for 8 weeks showed that the obesity of castrated Ldlr^−/− mice is independent of estrogens. Overall, our data reveal a novel role of Ldlr as functional modulator of metabolic alterations associated with hypogonadism.     

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis

The use of nicotinic acid to treat dyslipidemia is limited by induction of a “flushing” response, mediated in part by the interaction of prostaglandin D₂ (PGD₂) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr^−/−ApoE^−/− mice versus ApoE^−/− mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr^−/−ApoE^−/− mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE^−/− mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr^−/− mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.     

Hyperglycemic Ins2AkitaLdlr⁻/⁻ mice show severely elevated lipid levels and increased atherosclerosis: a model of type 1 diabetic macrovascular disease

Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2^AkitaLdlr^−/−). Ins2^AkitaLdlr^−/− mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr^−/− mice, 20-week-old Ins2^AkitaLdlr^−/− mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2^AkitaLdlr^−/− mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1β, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1β were also increased in the atherosclerotic lesions and artery walls of Ins2^AkitaLdlr^−/− mice. Thus, the Ins2^AkitaLdlr^−/− mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis.     

Osteoprotegerin Inhibits Aortic Valve Calcification and Preserves Valve Function in Hypercholesterolemic Mice

Background

There are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic Ldlr ^−/− Apob ^100/100 mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known.

Objectives

To determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic Ldlr ^−/− Apob ^100/100 mice.

Methods

Young Ldlr ^−/− Apob ^100/100 mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older Ldlr ^−/− Apob ^100/100 mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation.

Results

In Young Ldlr ^−/− Apob ^100/100 mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older Ldlr ^−/− Apob ^100/100 mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ∼80%, and attenuated aortic valve calcification by ∼ 70%. OPG also attenuated impairment of aortic valve function.

Conclusions

OPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone Ldlr ^−/− Apob ^100/100 mice.     

Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis

Background

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr^−/−Apobec1^−/−) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.

Methods/Principal Findings

In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr^−/−Apobec1^−/− mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10⁹ genome copies/mouse. Whereas Ldlr^−/−Apobec1^−/− mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr^−/−Apobec1^−/− mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.

Conclusions/Significance

Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.     

An Apolipoprotein A-I Mimetic Peptide Designed with a Reductionist Approach Stimulates Reverse Cholesterol Transport and Reduces Atherosclerosis in Mice

Apolipoprotein A-I (apoA-I) mimetic peptides are considered a promising novel therapeutic approach to prevent and/or treat atherosclerosis. An apoA-I mimetic peptide ELK-2A2K2E was designed with a reductionist approach and has shown exceptional activity in supporting cholesterol efflux but modest anti-inflammatory and anti-oxidant properties in vitro. In this study we compared these in vitro properties with the capacity of this peptide to modify rates of reverse cholesterol transport and development of atherosclerosis in mouse models. The peptide enhanced the rate of reverse cholesterol transport in C57BL/6 mice and reduced atherosclerosis in Apoe^−/− mice receiving a high fat diet. The peptide modestly reduced the size of the plaques in aortic arch, but was highly active in reducing vascular inflammation and oxidation. Administration of the peptide to Apoe^−/− mice on a high fat diet reduced the levels of total, high density lipoprotein and non-high density lipoprotein cholesterol and triglycerides. It increased the proportion of smaller HDL particles in plasma at the expense of larger HDL particles, and increased the capacity of the plasma to support cholesterol efflux. Thus, ELK-2A2K2E peptide reduced atherosclerosis in Apoe^−/− mice, however, the functional activity profile after chronic in vivo administration was different from that found in acute in vitro studies.     

Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner

Our laboratory previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knock-out mice (Ldlr^−/−xLcat^−/− or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to protection from the diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr^−/−xLcat^+/+ single-knock-out (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes, and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr^+/+xLcat^−/− (LCAT-KO) SC with DKO SC identified a role for LCAT deficiency in priming SC to express BAT genes. Additionally, we found that reduced cellular cholesterol is important for adipogenic differentiation, evidenced by increased induction of adipogenesis in cholesterol-depleted SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO mice is classical in origin, and its development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.     

Hyperlipidemia and atherosclerotic lesion development in Ldlr-deficient mice on a long-term high-fat diet

Background

Mice deficient in the LDL receptor (Ldlr ^−/− mice) have been widely used as a model to mimic human atherosclerosis. However, the time-course of atherosclerotic lesion development and distribution of lesions at specific time-points are yet to be established. The current study sought to determine the progression and distribution of lesions in Ldlr ^−/− mice.

Methodology/Principal Findings

Ldlr-deficient mice fed regular chow or a high-fat (HF) diet for 0.5 to 12 months were analyzed for atherosclerotic lesions with en face and cross-sectional imaging. Mice displayed significant individual differences in lesion development when fed a chow diet, whereas those on a HF diet developed lesions in a time-dependent and site-selective manner. Specifically, mice subjected to the HF diet showed slight atherosclerotic lesions distributed exclusively in the aortic roots or innominate artery before 3 months. Lesions extended to the thoracic aorta at 6 months and abdominal aorta at 9 months. Cross-sectional analysis revealed the presence of advanced lesions in the aortic sinus after 3 months in the group on the HF diet and in the innominate artery at 6 to 9 months. The HF diet additionally resulted in increased total cholesterol, LDL, glucose, and HBA1c levels, along with the complication of obesity.

Conclusions/Significance

Ldlr-deficient mice on the HF diet tend to develop site-selective and size-specific atherosclerotic lesions over time. The current study should provide information on diet induction or drug intervention times and facilitate estimation of the appropriate locations of atherosclerotic lesions in Ldlr ^−/− mice.     

Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.     

PI3K p110γ Deletion Attenuates Murine Atherosclerosis by Reducing Macrophage Proliferation but Not Polarization or Apoptosis in Lesions

Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR^−/−p110γ^+/− and LDLR^−/−p110γ^−/− mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ^+/− and p110γ^−/− mice. Lack of p110γ in LDLR^−/− mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR^−/−p110γ^−/− mice were smaller than in LDLR^−/−p110γ^+/− controls, which coincided with decreased macrophage proliferation in LDLR^−/−p110γ^−/− mouse lesions. This proliferation defect was also observed in p110γ^−/− bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR^−/−p110γ^−/− mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR^−/− mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.