Sex differences in atherosclerosis in mice with elevated phospholipid transfer protein activity are related to decreased plasma high density lipoproteins and not to increased production of triglycerides

Plasma phospholipid transfer protein (PLTP) has atherogenic properties in genetically modified mice. PLTP stimulates hepatic triglyceride secretion and reduces plasma levels of high density lipoproteins (HDL). The present study was performed to relate the increased atherosclerosis in PLTP transgenic mice to one of these atherogenic effects. A humanized mouse model was used which had decreased LDL receptor expression and was transgenic for human cholesterylester transfer protein (CETP) in order to obtain a better resemblance to the plasma lipoprotein profile present in humans. It is well known that female mice are more susceptible to atherosclerosis than male mice. Therefore, we compared male and female mice expressing human PLTP. The animals were fed an atherogenic diet and the effects on plasma lipids and lipoproteins, triglyceride secretion and the development of atherosclerosis were measured. The development of atherosclerosis was sex-dependent. This effect was stronger in PLTP transgenic mice, while PLTP activity levels were virtually identical. Also, the rates of hepatic secretion of triglycerides were similar. In contrast, plasma levels of HDL were about 2-fold lower in female mice than in male mice after feeding an atherogenic diet. We conclude that increased atherosclerosis caused by overexpression of PLTP is related to a decrease in HDL, rather than to elevated hepatic secretion of triglycerides.     

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.     

B cell deficiency progresses with lineage maturation in nude.X-linked immunodeficient mice B cell deficiency progresses with lineage maturation.

Previously we showed that unlike normal, nude, or X-linked immune deficient (xid) mice, nude.xid mice are deficient in bone marrow pre-B cell targets for Abelson murine leukemia virus transformation. We show that nude.xid bone marrow is deficient in both CD45(B220)+ and CD45(B220)- surface (s)IgM- progenitors that give rise to B cell colonies in Whitlock-Witte cultures. CD45(B220)+ precursors had normal differentiation potential in vitro. CD45(B220)- precursors differentiated into CD45(B220)+ cells at the same rate as normal controls, but acquired sIgM at a much slower rate. These results correlated with the observation that in nude.xid mice the severity of B lineage defects correlates with maturity: a profound (ninefold) deficit of sIgM+, CD45(B220)+ mature B cells, a fivefold deficit in the sIgM-, CD45(B220)+ precursors of short term B cell colonies (colonies forming within 4-5 days in Whitlock-Witte cultures), and a moderate (twofold) decrease in the frequency of sIgM-, CD45(B220)- (less mature) precursors of long term B cell colonies (colonies forming after 14 days of Whitlock-Witte culture. Thus the combination of the nude and xid mutations produces a deficiency in early B cell progenitors and the deficiency becomes more profound with further maturation. Therefore the lack of mature B cells is the result of a cascade effect. Inasmuch as bone marrow progenitors are affected, and these are the source of the vast majority of B cells, most B cells are affected by the xid mutation and the xid defect cannot be attributed to a loss of a fetal lineage of B cells. These results suggest that xid affected cells lack the capacity to progress efficiently through differentiation in the absence of an exogenous factor(s) that is dependent on the product of a normal allele at the nude locus. This product might be supplied in vivo by a T cell or T cell-dependent source and/or epithelial elements such as bone marrow stromal cells all of which are known to be affected by the nude mutation.     

Plasma coenzyme Q10 concentrations are not decreased in male patients with coronary atherosclerosis

Coenzyme Q₁₀ (CoQ₁₀) is an important mitochondrial electron transfer component and has been postulated to function as a powerful antioxidant protecting LDL from oxidative damage. It could thus reduce the risk of cardiovascular disease. Thus far, beneficial effects of supplementation with CoQ₁₀ have been reported. To study the relation between unsupplemented concentrations of plasma CoQ₁₀ and coronary atherosclerosis, we performed a case-control study among 71 male cases with angiographically documented severe coronary atherosclerosis and 69 healthy male controls free from symptomatic cardiovascular disease and without atherosclerotic plaques in the carotid artery.

Plasma CoQ₁₀ concentrations (mean ± SE) were 0.86 ± 0.04 vs. 0.83 ± 0.04 μmol/l for cases and controls, respectively. The CoQ₁₀/LDL-cholesterol ratio (μmol/mmol) was slightly lower in cases than in controls (0.22 ± 0.01 vs. 0.26 ± 0.03). Differences in CoQ₁₀ concentrations and CoQ₁₀/LDL-cholesterol ratio did not reach significance. The odds ratios (95% confidence interval) for the risk of coronary atherosclerosis calculated per μmol/l increase of CoQ₁₀ was 1.12 (0.28-4.43) after adjustment for age, smoking habits, total cholesterol and diastolic blood pressure.

We conclude that an unsupplemented plasma CoQ₁₀ concentration is not related to risk of coronary atherosclerosis.     

Plasma coenzyme Q10 concentrations are not decreased in male patients with coronary atherosclerosis

Coenzyme Q₁₀ (CoQ₁₀) is an important mitochondrial electron transfer component and has been postulated to function as a powerful antioxidant protecting LDL from oxidative damage. It could thus reduce the risk of cardiovascular disease. Thus far, beneficial effects of supplementation with CoQ₁₀ have been reported. To study the relation between unsupplemented concentrations of plasma CoQ₁₀ and coronary atherosclerosis, we performed a case-control study among 71 male cases with angiographically documented severe coronary atherosclerosis and 69 healthy male controls free from symptomatic cardiovascular disease and without atherosclerotic plaques in the carotid artery.

Plasma CoQ₁₀ concentrations (mean ± SE) were 0.86 ± 0.04 vs. 0.83 ± 0.04 μmol/l for cases and controls, respectively. The CoQ₁₀/LDL-cholesterol ratio (μmol/mmol) was slightly lower in cases than in controls (0.22 ± 0.01 vs. 0.26 ± 0.03). Differences in CoQ₁₀ concentrations and CoQ₁₀/LDL-cholesterol ratio did not reach significance. The odds ratios (95% confidence interval) for the risk of coronary atherosclerosis calculated per μmol/l increase of CoQ₁₀ was 1.12 (0.28–4.43) after adjustment for age, smoking habits, total cholesterol and diastolic blood pressure.

We conclude that an unsupplemented plasma CoQ₁₀ concentration is not related to risk of coronary atherosclerosis.     

Apolipoprotein B production and very low density lipoprotein secretion by calf liver slices.

Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [35S]methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo B but rather to a defect in VLDL assembly and/or secretion.     

Both Serum Apolipoprotein B and the Apolipoprotein B/Apolipoprotein A-I Ratio Are Associated with Carotid Intima-Media Thickness

Background

Previous studies indicated that apolipoprotein measurements predicted cardiovascular disease (CVD) risk; however, associations between apolipoproteins and carotid intima-media thickness (CIMT) were less explored.

Methodology and Principal Findings

The cross-sectional study included 6069 participants aged 40 years or older with NGT from Shanghai, China. Serum fasting traditional lipids (total cholesterol [TC], low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol [HDL-C] and triglycerides [TG]), apoA-I and apoB were assessed. A high-resolution B-mode ultrasonography was performed to measure CIMT. We found CIMT increased progressively across the quartiles of serum apoB (p for trend <0.0001). In logistic regression, concentrations of apoB (odds ratio [OR] 1.27, 95% confidence interval [CI] 1.18–1.36), TC (OR 1.23, 95% CI 1.14–1.32), LDL-C (OR 1.25, 95% CI 1.16–1.34) and TG (OR 1.11, 95% CI 1.04–1.20) were significantly related to elevated CIMT after adjusted for age and sex. Meanwhile, the apoB/apoA-I ratio (OR 1.25, 95% CI 1.17–1.34) related to elevated CIMT. ApoB (OR 1.23, 95% CI 1.00–1.51) and the apoB/apoA-I ratio (OR 1.19, 95% CI 1.04–1.36) remained significantly associated with elevated CIMT, after adjusted for the traditional CVD risk factors including traditional lipids.

Conclusions and Significance

There were significant associations between serum apoB, the apoB/apoA-I ratio and elevated CIMT. Serum apoB and the apoB/apoA-I ratio might be independent predictors of early atherosclerosis in NGT.     

A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice

KK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice. Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins.     

Dietary protein deficiency in pregnant mice and offspring

Previous studies suggest an association between dermal contact hypersensitivity and preterm delivery. We hypothesized that dietary protein deficiency produces cell-mediated immune hypersensitivity in pregnant animals and their offspring akin to those known to produce tissue damage. We compared the effects of feeding a 20% protein diet (controls) to those of feeding a 10% protein (deficient) diet ad libitum to pregnant BALB/c mice. We measured dermal contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) by the increment in ear skin thickness (swelling) 72 h after immunization and parity by the number of viable pups delivered. Dams fed the protein-deficient diet ingested less food, gained less weight and delivered fewer viable pups than the dams fed the control diet. Greater DNFB-stimulated increment in ear skin thickness was found in the protein-deficient mothers and in their offspring than in the control mothers and their offspring. We conclude that dietary protein deficiency limits parity and induces immune hypersensitivity. These findings suggest the potential for dietary protein deficiency to activate a T-cell-mediated branch of the immune response that may put pregnant animals at risk for preterm delivery.     

Akt3 deficiency in macrophages promotes foam cell formation and atherosclerosis in mice

Akt, a serine-threonine protein kinase, exists as three isoforms. The Akt signaling pathway controls multiple cellular functions in the cardiovascular system, and the atheroprotective endothelial cell-dependent role of Akt1 has been recently demonstrated. The role of Akt3 isoform in cardiovascular pathophysiology is not known. We explored the role of Akt3 in atherosclerosis using mice with a genetic ablation of the Akt3 gene. Using hyperlipidemic ApoE(-/-) mice, we demonstrated a macrophage-dependent, atheroprotective role for Akt3. In vitro experiments demonstrated differential subcellular localization of Akt1 and Akt3 in macrophages and showed that Akt3 specifically inhibits macrophage cholesteryl ester accumulation and foam cell formation, a critical early event in atherogenesis. Mechanistically, Akt3 suppresses foam cell formation by reducing lipoprotein uptake and promoting ACAT-1 degradation via the ubiquitin-proteasome pathway. These studies demonstrate the nonredundant atheroprotective role for Akt3 exerted via the previously unknown link between the Akt signaling pathway and cholesterol metabolism.     

Apolipoprotein E: from atherosclerosis to Alzheimer's disease and beyond.

Apolipoprotein E is a key regulator of plasma lipid levels. Our appreciation of its role continues to expand as additional aspects of its function are discovered. Apolipoprotein E affects the levels of all lipoproteins, either directly or indirectly by modulating their receptor-mediated clearance or lipolytic processing and the production of hepatic very low density lipoproteins. Furthermore, it plays a critical role in neurobiology. The apolipoprotein E4 allele is the major susceptibility gene related to the occurrence and early age of onset of Alzheimer's disease. It is probable that one of the major functions of apolipoprotein E in the central nervous system is to mediate neuronal repair, remodeling, and protection, with apolipoprotein E4 being less effective than the E3 and E2 alleles. The isoform-specific effects of apolipoprotein E are currently being unraveled through detailed structure and function studies of this protein.     

NOX1 deficiency in apolipoprotein E-knockout mice is associated with elevated plasma lipids and enhanced atherosclerosis

Abstract

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE^?/?) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE^?/? versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE^?/? mice. To examine the impact of a further reduction in NOX1 activity, APOE^?/? mice were crossed with NOX1^?/y mice to generate NOX1^?/y/APOE^?/? double-knockouts. NOX1 deficiency in APOE^?/? mice was associated with 30–50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1^?/y/APOE^?/? versus APOE^?/? mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1^?/y/APOE^?/? and APOE^?/? mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE^?/? mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE^?/? mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.     

Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice

Previous studies with hypertriglyceridemic APOC3 transgenic mice have suggested that apolipoprotein C-III (apoC-III) may inhibit either the apoE-mediated hepatic uptake of TG-rich lipoproteins and/or the lipoprotein lipase (LPL)-mediated hydrolysis of TG. Accordingly, apoC3 knockout (apoC3(-/-)) mice are hypotriglyceridemic. In the present study, we attempted to elucidate the mechanism(s) underlying these phenomena by intercrossing apoC3(-/-) mice with apoE(-/-) mice to study the effects of apoC-III deficiency against a hyperlipidemic background. Similar to apoE(+/+) apoC3(-/-) mice, apoE(-/-)apoC3(-/-) mice exhibited a marked reduction in VLDL cholesterol and TG, indicating that the mechanism(s) by which apoC-III deficiency exerts its lipid-lowering effect act independent of apoE. On both backgrounds, apoC3(-/-) mice showed normal intestinal lipid absorption and hepatic VLDL TG secretion. However, turnover studies showed that TG-labeled emulsion particles were cleared much more rapidly in apoC3(-/-) mice, whereas the clearance of VLDL apoB, as a marker for whole particle uptake by the liver, was not affected. Furthermore, it was shown that cholesteryl oleate-labeled particles were also cleared faster in apoC3(-/-) mice. Thus the mechanisms underlying the hypolipidemia in apoC3(-/-) mice involve both a more efficient hydrolysis of VLDL TG as well as an enhanced selective clearance of VLDL cholesteryl esters from plasma. In summary, our studies of apoC3(-/-) mice support the concept that apoC-III is an effective inhibitor of VLDL TG hydrolysis and reveal a potential regulating role for apoC-III with respect to the selective uptake of cholesteryl esters.     

Streptozotocin-induced diabetes in human apolipoprotein B transgenic mice. Effects on lipoproteins and atherosclerosis

The effects of diabetes and lipoprotein lipase (LpL) on plasma lipids were studied in mice expressing human apolipoprotein B (HuBTg). Our overall objective was to produce a diabetic mouse model in which the sole effects of blood glucose elevation on atherosclerosis could be assessed. Mice were made diabetic by intraperitoneal injection of streptozotocin, which led to a 2- to 2. 5-fold increase in plasma glucose. Lipids were assessed in mice on chow and on an atherogenic Western type diet (WTD), consisting of 21% (wt/wt) fat and 0.15% (wt/wt) cholesterol. Plasma triglyceride and cholesterol were the same in diabetic and non-diabetic mice on the chow diet. On the WTD, male diabetic HuBTg mice had a >50% increase in plasma cholesterol and more very low density lipoprotein (VLDL) cholesterol and triglyceride as assessed by FPLC analysis. A Triton study showed no increase in triglyceride or apolipoprotein B production, suggesting that the accumulation of VLDL was due to a decrease in lipoprotein clearance. Surprisingly, the VLDL increase in these mice was not due to a decrease in LpL activity in postheparin plasma. To test whether LpL overexpression would alter these diabetes-induced lipoprotein changes, HuBTg mice were crossed with mice expressing human LpL in muscle. LpL overexpression reduced plasma triglyceride, but not cholesterol, in male mice on WTD. Aortic root atherosclerosis assessed in 32-week-old mice on the WTD was not greater in diabetic mice. In summary, diabetes primarily increased plasma VLDL in HuBTg mice. LpL activity was not decreased in these animals. However, additional LpL expression eliminated the diabetic lipoprotein changes. These mice did not have more atherosclerosis with diabetes.     

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.     

B cells of aged mice show decreased expansion in response to antigen,but are normal in effector function

Increased dysfunction of the immune system with age can be attributed to developmental changes in cell types critical for proper immune responses. Previous studies have shown defects in humoral responses of aged individuals, but have not distinguished between aged T-cell/microenvironment and intrinsic B-cell defects. Here adoptive transfer of antigen-specific transgenic B cells compared early immunopoeisis from young and aged donors in a young recipient environment. B cells from aged donors demonstrated decreased antigen-induced expansion, particularly in the lymph nodes; however, they acquired a germinal center phenotype at frequencies similar to B cells from young donors. Additionally, aged B cells produced equivalent levels of antigen-specific antibody that underwent affinity maturation and isotype switching and demonstrated similar numbers of antibody-secreting cells of switched isotype. Thus, the ability of aged B cells to respond appropriately to T-dependent antigens and differentiate into high-affinity, isotype-switched, antibody-secreting cells appears to be intact.