High Plasma Cholesterol Research Articles

Epistatic Interactions between Apolipoprotein E and Hemoglobin S Genes in Regulation of Malaria Parasitemia

Apolipoprotein E is a monomeric protein secreted by the liver and responsible for the transport of plasma cholesterol and triglycerides. The APOE gene encodes 3 isoforms Ɛ4, Ɛ3 and Ɛ2 with APOE Ɛ4 associated with higher plasma cholesterol levels and increased pathogenesis in several infectious diseases (HIV, HSV). Given that cholesterol is an important nutrient for malaria parasites, we examined whether APOE Ɛ4 was a risk factor for Plasmodium infection, in terms of prevalence or parasite density. A cross sectional survey was performed in 508 children aged 1 to 12 years in Gabon during the wet season. Children were screened for Plasmodium spp. infection, APOE and hemoglobin S (HbS) polymorphisms. Median parasite densities were significantly higher in APOE Ɛ4 children for Plasmodium spp. densities compared to non-APOE Ɛ4 children. When stratified for HbS polymorphisms, median Plasmodium spp. densities were significantly higher in HbAA children if they had an APOE Ɛ4 allele compared to those without an APOE Ɛ4 allele. When considering non-APOE Ɛ4 children, there was no quantitative reduction of Plasmodium spp. parasite densities for HbAS compared to HbAA phenotypes. No influence of APOE Ɛ4 on successful Plasmodium liver cell invasion was detected by multiplicity of infection. These results show that the APOE Ɛ4 allele is associated with higher median malaria parasite densities in children likely due to the importance of cholesterol availability to parasite growth and replication. Results suggest an epistatic interaction between APOE and HbS genes such that sickle cell trait only had an effect on parasite density in APOE Ɛ4 children. This suggests a linked pathway of regulation of parasite density involving expression of these genes. These findings have significance for understanding host determinants of regulation of malaria parasite density, the design of clinical trials as well as studies of co-infection with Plasmodium and other pathogens.

Inhibition of Nuclear Factor of Activated T-Cells (NFAT) Suppresses Accelerated Atherosclerosis in Diabetic Mice

Objective of the StudyDiabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis.Methodology and Principal FindingsStreptozotocin (STZ)-induced diabetes in apolipoprotein E−/− mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice.ConclusionsTargeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications.

176 CONTRACTILITY IS INCREASED IN THORACIC AORTAE OF APOLIPOPROTEIN E KNOCK-OUT MICE PRIOR TO LESION DEVELOPMENT

High plasma cholesterol has been implicated as a risk factor in the development of atherosclerosis and hypertension. The removal of cholesterol from cellular membranes using methyl-β-cyclodextrin has been shown to reduce contractility in the vascular smooth muscle in response to a range of agonists. The mechanism for this has been linked to the disruption of caveolae and the associated signalling micro-domains. In particular it has been shown that disruption of caveolae can reduce store-operated calcium entry which, in some vascular tissues, can contribute to contraction. There is currently limited evidence showing the effects of high serum cholesterol on vascular function. In the current study, thoracic aorta contractility was assessed in apolipoprotein E knock-out (ApoE−/−) and control (C57/Bl6) mice fed a standard chow diet for 8 weeks. Contraction was stimulated through depolarisation (100mM KCl) and the use of the agonists phenylephrine (PE; 10 µM) and serotonin (5-HT; 10 µM), on vessel segments mounted on a wire myograph. In addition, fluorescence microscopy was employed to measure global intracellular calcium concentrations in INDO-1 (20 µM) loaded segments of thoracic aorta. Store operated calcium entry was induced by removal of [Ca2+]e and incubation with cyclopiazonic acid (CPA; 10 µM) followed by re-introduction of calcium (2mM). Data are represented as means±S.E.M., compared using one-way ANOVA. In response to a depolarising stimulus, there was no change in contractility observed between chow fed control and ApoE−/− mice (1.98±0.17 mN vs 2.08±0.20 mN increase in tension; n=11,4, respectively). However, contractility in chow fed ApoE−/− mice when compared to the chow fed age and strain matched controls was significantly increased after exposure to both phenylephrine (47.2±5.5 vs. 82.1±9.7% normalised contraction; p

Abstract 477: HDL Inhibition of Murine Atherosclerosis Lesion Development is Dependent on Strain Background

Inbred mouse strains vary greatly in their relative atherosensitivity, with C57BL/6 (C57) being the most sensitive and hence the preferred strain for most atherosclerosis studies. Among the resistant strains, FVBN/J (FVB) has been relatively well studied. On the ApoE knockout (E°) background, both mouse strains develop atherosclerotic lesions which are exacerbated by feeding a high fat/high cholesterol western-type diet (WTD). However, the FVB-E° mouse exhibits nearly 10-fold smaller lesions than the C57-E°. Additionally, FVB mice have plasma HDL cholesterol levels that are twice as high as C57. Wild type FVB mice have been shown to have lower inflammatory cell recruitment to the peritoneum upon thioglycollate treatment than C57. Our lab has generated ApoE/ApoA-I double knockout mice (EA°, lacking HDL) on both C57 and FVB background to compare the effect of HDL deficiency on lesion development. We hypothesize that the loss of HDL will have a greater effect on the more lesion susceptible C57 than the resistant FVB. 8 week C57 and FVB E° and EA° mice were kept on a WTD for 6 or 10 weeks and lesions were quantified in the innominate artery, ascending aorta, and aortic root. In all cases mice in the C57 background displayed significantly greater lesion size than mice in the FVB background, except in the ascending aorta of the E° mice at the earlier time point, despite higher plasma cholesterol levels in the FVB mice. The presence of ApoA-I was protective (E° vs EA°) in the C57 strain in the ascending aorta at both time points and its protection became significant in the innominate artery at the 10 week time point. No additional protection was afforded by ApoA-I in the FVB strain. ApoA-I deficiency in FVB mice did not affect inflammatory cell recruitment to the peritoneal cavity or plasma markers of LDL oxidation as greatly as in C57 mice. Plasma monocyte counts both before and after 6 weeks on WTD were higher in C57 mice with no effect of ApoA-I in either strain. In conclusion, the FVB strain is less sensitive than C57 to lesion development at 3 major arterial sites with no protection afforded by HDL/ApoA-I, whereas HDL/ApoA-I is significantly protective in the C57 strain. This protection may result from the anti-inflammatory/anti-oxidative nature of C57 HDL that is not observed in FVB mice.

Extensive Xanthomas and Severe Subclinical Atherosclerosis in Homozygous Familial Hypercholesterolemia

![Figure][1] An asymptomatic 20-year-old man presented with a history of high plasma cholesterol levels and gigantic tendinous xanthomas over the extensor tendons of his metacarpophalangeal joints and patellas (A to C) . Laboratory tests showed total cholesterol of 785 mg/dl, low-density

Effects of lifestyle modifications on cognitive impairments in a mouse model of hypercholesterolemia

Epidemiological studies indicate that high midlife plasma cholesterol levels increases the risk of Alzheimer's disease. Moreover, middle-aged familial hypercholesterolemia (FH) subjects show a particularly high incidence of mild cognitive impairments (MCI). These evidence points to hypercholesterolemia as one of the modifiable risk factors focused on prevention/treatment of cognitive deterioration. The present study draws a comparison between pharmacological (lipid-lowering drug probucol) and non-pharmacological (voluntary running wheel, RW) approaches for the management of hypercholesterolemia and cognitive impairments associated with the low-density lipoprotein receptor-deficient (LDLr−/−) mice, a well-established rodent model of FH. We also investigated whether exposure to environmental enrichment (EE), a feasible option to increase physical activity in young mice cohort, from birth to adolescence (PN45) yields long-term behavioral changes in adult LDLr−/− mice (PN90). We observed that both probucol and RW significantly decreased total and non-HDL plasma cholesterol levels in LDLr−/− mice. Notably, only physical exercise mitigated the spatial memory deficits of LDLr−/− mice. In addition, we showed that exposure to EE from birth until the adolescence did not mitigate the spatial memory deficits of adult LDLr−/− mice in the object location task, although it induced persistent anxyolitic-like effects in the open field arena. Collectively, our results emphasize the advantages physical exercise, in comparison to lipid-lowering drugs, for the management of cognitive deficits associated with FH.

Diets for body weight control and health: the potential of changing the macronutrient composition

At the beginning of the last century obesity and type 2 diabetes were treated quite successfully using low-carbohydrate diets. Following the discovery of insulin, the carbohydrate content of the diabetic diet became more liberal, as glycaemia and glycosuria could be controlled, more or less well, with hypoglycaemic medication and insulin treatment. Later, saturated fats and high-plasma cholesterol concentrations were implicated in cardiovascular disease and since then high-carbohydrate diets have become synonymous with 'health' and have been conventional nutrition doctrine for the past 40 years. In spite of this, the prevalence of some non-communicable metabolic diseases have increased to epidemic proportions and have led an increasing number of researchers in the fields of medicine and nutrition to challenge the validity of present-day dietary guidelines. There is increasing evidence that diets with a lower, or even very-low, carbohydrate content can help overweight and obese individuals to lose and maintain lost weight, diabetics to control blood glucose with more ease and prevent the development of diabetic complications, while at the same time improving blood lipid profiles and biomarkers of cardiovascular risk. The present review considers the evolution of our diet and questions whether high-carbohydrate diets are indeed synonymous with health.

DETERMINING THE FOLDING DOMAIN OF LIPID FREE APOA-I IN SOLUTION BY MUTATION†

DETERMINING THE FOLDING DOMAIN OF LIPID FREE APOA-I IN SOLUTION BY MUTATION†

Pharmacodynamic and pharmacokinetic analysis of apoE4 [L261A, W264A, F265A, L268A, V269A], a recombinant apolipoprotein E variant with improved biological properties

Physiological levels of wild-type (wt) apolipoprotein E (apoE) in plasma mediate the clearance of cholesterol-rich atherogenic lipoprotein remnants while higher than normal plasma apoE concentrations fail to do so and trigger hypertriglyceridemia. This property of wt apoE reduces significantly its therapeutic value as a lead biological for the treatment of dyslipidemia. Recently, we reported the generation of a recombinant apoE variant, apoE4 [L261A, W264A, F265A, L268A, V269A] (apoE4mut1) with improved biological functions. Specifically, in apoE-deficient (apoE−/−) mice this variant can normalize high plasma cholesterol levels without triggering hypertriglyceridemia, even at supraphysiological levels of expression. In the present study we performed pharmacodynamic and pharmacokinetic analysis of apoE4mut1 in experimental mice. Using adenovirus-mediated gene transfer in LDL receptor deficient (LDLr−/−) mice, we show that the cholesterol lowering potential of apoE4mut1 is dependent on the expression of a functional classical LDLr. Bolus infusion of apoE4mut1-containing proteoliposomes in apoE−/− mice fed western-type diet for 6 weeks indicated that exogenously synthesized apoE4mut1 maintains intact its ability to normalize the high cholesterol levels of these mice with a maximum pharmacological effect obtained at 10h post-treatment. Interestingly, plasma cholesterol levels remained significantly reduced up to 24h following intravenous administration of apoE4mut1 proteoliposomes. Measurements of plasma apoE levels indicated that apoE4mut1 in the form of proteoliposomes used in the study has a half-life of 15.8h. Our data suggest that purified apoE4mut1 may be an attractive new candidate for the acute correction of hypercholesterolemia in subjects expressing functional LDL receptor.

Evaluation of Chlamydophila psittaci infection and other risk factors for atherosclerosis in pet psittacine birds

To determine whether the presence of Chlamydophila psittaci antigen, plasma cholesterol concentration, diet, sex, species, and age are risk factors for the development of atherosclerosis in pet psittacine birds. Retrospective case-control study. 31 psittacine birds with atherosclerosis (study birds) and 31 psittacine birds without atherosclerosis (control birds). Necropsy reports were reviewed, birds with a histopathologic diagnosis of atherosclerosis were identified, and available medical records were reviewed. Signalment, history, clinicopathologic findings, and other relevant data were recorded and evaluated. Control birds did not have atherosclerosis and were chosen by both convenience sampling and population demographics. Histologic sections of great vessels from all birds (study and control birds) were reviewed and then submitted for immunohistochemical staining for the presence of C psittaci antigen. Result of immunohistochemical staining for C psittaci antigen in blood vessels was significantly associated with atherosclerosis. After adjusting for age, species origin, and type of illness, the odds of atherosclerosis was 7 times as high for birds with positive immunohistochemical staining for C psittaci antigen, compared with that of birds with negative immunohistochemical staining. Study birds and control birds differed significantly only with respect to plasma cholesterol concentrations. The median plasma cholesterol concentration of study birds (421 mg/dL) was significantly higher than that of control birds (223 mg/dL). Infection with C psittaci and a high plasma cholesterol concentration may be risk factors for developing atherosclerosis in pet psittacine birds.

Serum adiponectin level is not only decreased in metabolic syndrome but also in borderline metabolic abnormalities

Objective:Along with the increasing prevalence of obesity and related diseases, particularly atherosclerotic diseases, metabolic syndrome (MetS) is now a common and major public health issue in many countries around the world. Adiponectin, a protein secreted by the adipose tissue, has become recognized as a key player in the development of MetS. These days, not only MetS but also borderline metabolic/physiological abnormalities, such as impaired fasting glucose, high normal blood pressure and high normal plasma cholesterol, have been reported to be risk factors for atherosclerotic disease. Therefore, we undertook this study to determine the relationship between adiponectin and borderline metabolic/physiological abnormalities, as well as MetS.Design:A cross-sectional study performed from April 2007 to November 2009.Subjects:In 16 892 Japanese adults (10 008 men and 6884 women), we examined the relationship between the serum adiponectin concentration and borderline metabolic/physiological abnormalities or MetS by a questionnaire survey about medical treatment, body size measurement and measurement of laboratory parameters including the serum adiponectin concentration.Results:Adiponectin showed a significant negative correlation with the number of MetS components. In subjects without overt diabetes mellitus, hypertension or dyslipidemia, the adiponectin concentration also showed a significant negative correlation with the number of borderline metabolic abnormalities.Conclusion:The decrease of circulating adiponectin may start before the development of diabetes mellitus, hypertension, dyslipidemia or MetS. Adiponectin is an important biomarker for reflecting the adverse influence of visceral fat in persons with MetS, and also in these subclinical states.

Development of Apolipoprotein E–Deficient Mice

The first mouse carrying a deletion mutation in its apolipoprotein E (apoE) gene was born in our mouse colony in the summer of 1991. Our ongoing experiments to that point had indicated that this would happen, but the realization that the mouse in front of my eyes had inherited a mutation that we had designed and made in the laboratory filled me with awe. It did not occur to me immediately that this mouse would develop hypercholesterolemia and atherosclerosis and dictate the direction of my research for the next 20 years. I here briefly describe the background and my personal experience that led to the development of apoE-deficient mice as a model for human atherosclerosis. As a research scientist in the Department of Genetics at the University of Wisconsin, my main interest during the early 1980s was directed toward understanding how different types of DNA recombination contribute to the architecture and diversity of individual genomes within human populations and to DNA differences between humans and nonhuman primates. My studies clearly showed that new alleles and new genes are constantly being formed in the human genome by recombinational events, particularly between existing members of multigene families. There also appeared to be hot spots for recombination. By tracing a history of the human haptoglobin-gene cluster from the present-day structures in human populations and in other primates, I found that a single recombination event between related genes often produces much more drastic changes in the genome than those that result from simple point mutations.1 Whether this frequent yet natural occurrence of homologous recombination in the mammalian genome could be used to alter the genome of mammalian cells in culture in a preplanned fashion became a question of interest to several laboratories.2,3 An affirmative answer was first obtained in 1985, …

Side Chain-oxidized Oxysterols Regulate the Brain Renin-Angiotensin System through a Liver X Receptor-dependent Mechanism

Disturbances in cholesterol metabolism have been associated with hypertension and neurodegenerative disorders. Because cholesterol metabolism in the brain is efficiently separated from plasma cholesterol by the blood-brain barrier (BBB), it is an unsolved paradox how high blood cholesterol can cause an effect in the brain. Here, we discuss the possibility that cholesterol metabolites permeable to the BBB might account for these effects. We show that 27-hydroxycholesterol (27-OH) and 24S-hydroxycholesterol (24S-OH) up-regulate the renin-angiotensin system (RAS) in the brain. Brains of mice on a cholesterol-enriched diet showed up-regulated angiotensin converting enzyme (ACE), angiotensinogen (AGT), and increased JAK/STAT activity. These effects were confirmed in in vitro studies with primary neurons and astrocytes exposed to 27-OH or 24S-OH, and were partially mediated by liver X receptors. In contrast, brain RAS activity was decreased in Cyp27a1-deficient mice, a model exhibiting reduced 27-OH production from cholesterol. Moreover, in humans, normocholesterolemic patients with elevated 27-OH levels, due to a CYP7B1 mutation, had markers of activated RAS in their cerebrospinal fluid. Our results demonstrate that side chain-oxidized oxysterols are modulators of brain RAS. Considering that levels of cholesterol and 27-OH correlate in the circulation and 27-OH can pass the BBB into the brain, we suggest that this cholesterol metabolite could be a link between high plasma cholesterol levels, hypertension, and neurodegeneration.

Angiotensin type 1 receptor modulates macrophage polarization and renal injury in obesity

The mechanisms for increased risk of chronic kidney disease (CKD) in obesity remain unclear. The renin-angiotensin system is implicated in the pathogenesis of both adiposity and CKD. We investigated whether the angiotensin type 1 (AT(1)) receptor, composed of dominant AT(1a) and less expressed AT(1b) in wild-type (WT) mice, modulates development and progression of kidney injury in a high-fat diet (HFD)-induced obesity model. WT mice had increased body weight, body fat, and insulin levels and decreased adiponectin levels after 24 wk of a high-fat diet. Identically fed AT(1a) knockout (AT1aKO) mice gained weight similarly to WT mice, but had lower body fat and higher plasma cholesterol. Both obese AT1aKO and obese WT mice had increased visceral fat and kidney macrophage infiltration, with more proinflammatory M1 macrophage markers as well as increased mesangial expansion and tubular vacuolization, compared with lean mice. These abnormalities were heightened in the obese AT1aKO mice, with downregulated M2 macrophage markers and increased macrophage AT(1b) receptor. Treatment with an AT(1) receptor blocker, which affects both AT(1a) and AT(1b), abolished renal macrophage infiltration with inhibition of renal M1 and upregulation of M2 macrophage markers in obese WT mice. Our data suggest obesity accelerates kidney injury, linked to augmented inflammation in adipose and kidney tissues and a proinflammatory shift in macrophage and M1/M2 balance.

Myths and Facts Concerning the Use of Statins in Very Old Patients

As population grows old, the number of persons at risk of cardiovascular events also grows. Though octogenarians form a small percentage of the general population their absolute risk of coronary and cerebrovascular disease is high, but there is still some doubt as to whether high plasma cholesterol levels increase vascular risk in this age group, as published data are conflicting. There is evidence that elevated plasma cholesterol increases the risk of coronary artery disease in older adults, and an inverse linear relationship was found between HDL cholesterol levels and the risk of mortality from ischemic heart disease in all age groups. The relationship between total plasma cholesterol and the risk of death from ischemic stroke is weak in younger populations and is even lower in people between 70 and 89 years, and is inverse for hemorrhagic stroke. However, studies showed that statin treatment lowers the risk of ischemic stroke, independently of age. Statins are underused in the elderly, perhaps because of lack of perception of the real vascular risk of older adults, concerns about statin efficacy or safety in this population, or the increase of comorbidities and polypharmacy which could affect adherence to drug-treatment. Trials designed to address this issues are urgently needed, in order to be able to make evidence-guided decisions on lipid management of the elderly.

Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4

We have used adenovirus-mediated gene transfer in apolipoprotein (apo)E(-/-) mice to elucidate the molecular etiology of a dominant form of type III hyperlipoproteinemia (HLP) caused by the R142C substitution in apoE4. It was found that low doses of adenovirus expressing apoE4 cleared cholesterol, whereas comparable doses of apoE4[R142C] greatly increased plasma cholesterol, triglyceride, and apoE levels, caused accumulation of apoE in VLDL/IDL/LDL region, and promoted the formation of discoidal HDL. Co-expression of apoE4[R142C] with lecithin cholesterol acyltransferase (LCAT) or lipoprotein lipase (LPL) in apoE(-/-) mice partially corrected the apoE4[R142C]-induced dyslipidemia. High doses of C-terminally truncated apoE4[R142C]-202 partially cleared cholesterol in apoE(-/-) mice and promoted formation of discoidal HDL. The findings establish that apoE4[R142C] causes accumulation of apoE in VLDL/IDL/LDL region and affects in vivo the activity of LCAT and LPL, the maturation of HDL, and the clearance of triglyceride-rich lipoproteins. The prevention of apoE4[R142C]-induced dyslipidemia by deletion of the 203-299 residues suggests that, in the full-length protein, the R142C substitution may have altered the conformation of apoE bound to VLDL/IDL/LDL in ways that prevent triglyceride hydrolysis, cholesterol esterification, and receptor-mediated clearance in vivo.

Hypercholesterolemia and microvascular dysfunction: interventional strategies

Hypercholesterolemia is defined as excessively high plasma cholesterol levels, and is a strong risk factor for many negative cardiovascular events. Total cholesterol levels above 200 mg/dl have repeatedly been correlated as an independent risk factor for development of peripheral vascular (PVD) and coronary artery disease (CAD), and considerable attention has been directed toward evaluating mechanisms by which hypercholesterolemia may impact vascular outcomes; these include both results of direct cholesterol lowering therapies and alternative interventions for improving vascular function. With specific relevance to the microcirculation, it has been clearly demonstrated that evolution of hypercholesterolemia is associated with endothelial cell dysfunction, a near-complete abrogation in vascular nitric oxide bioavailability, elevated oxidant stress, and the creation of a strongly pro-inflammatory condition; symptoms which can culminate in profound impairments/alterations to vascular reactivity. Effective interventional treatments can be challenging as certain genetic risk factors simply cannot be ignored. However, some hypercholesterolemia treatment options that have become widely used, including pharmaceutical therapies which can decrease circulating cholesterol by preventing either its formation in the liver or its absorption in the intestine, also have pleiotropic effects with can directly improve peripheral vascular outcomes. While physical activity is known to decrease PVD/CAD risk factors, including obesity, psychological stress, impaired glycemic control, and hypertension, this will also increase circulating levels of high density lipoprotein and improving both cardiac and vascular function. This review will provide an overview of the mechanistic consequences of the predominant pharmaceutical interventions and chronic exercise to treat hypercholesterolemia through their impacts on chronic sub-acute inflammation, oxidative stress, and microvascular structure/function relationships.

Apolipoprotein A-I Deficiency Increases Cerebral Amyloid Angiopathy and Cognitive Deficits in APP/PS1ΔE9 Mice

A hallmark of Alzheimer disease (AD) is the deposition of amyloid β (Aβ) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aβ(40) aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Aβ(42) and decreases Aβ(42) toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1ΔE9 to apoA-I(KO) mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1ΔE9 mice. Further characterization of APP/PS1ΔE9/apoA-I(KO) mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble Aβ oligomer levels, Aβ plaque load, or levels of insoluble Aβ in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aβ isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I(KO) mice, insoluble Aβ(40) is increased more than 10-fold, and Aβ(42) is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1ΔE9/apoA-I(KO) mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased Aβ toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1ΔE9 mice in parallel to significantly increased cerebral amyloid angiopathy.

Effects of fat and rumen-protected methionine supplementation on production response of first-lactation cows

To determine whether supplementation of rumen-protected methionine (RPM) and fat, from 0 to 150 days in milk, improves milk production, milk quality, and energy balance (EB), in a completely randomized design, 32 first-lactation cows fed diets with 0 or 3.5% added fat and 0 or 0.15% added RPM. Fat, RPM, and fat plus RPM increased milk yield. Supplemental RPM increased milk protein (%) and decreased milk fat (%). Fat, RPM, and fat plus RPM increased (kg) milk fat, milk protein, and milk lactose. Cows fed fat and RPM alone had greater negative EB values than cows non-supplemented. Cows fed fat plus RPM had similar EB to cows non-supplemented. Supplemental RPM induced higher plasma glucose and lower urea-N. Fat induced higher plasma cholesterol. In conclusion, supplemental fat or RPM, and fat plus RPM increased milk production, but fat plus RPM enhanced EB as compared to fat or RPM alone.

Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia

Whole-genome sequencing is a potentially powerful tool for the diagnosis of genetic diseases. Here, we used sequencing-by-ligation to sequence the genome of an 11-month-old breast-fed girl with xanthomas and very high plasma cholesterol levels (1023 mg/dl). Her parents had normal plasma cholesterol levels and reported no family history of hypercholesterolemia, suggesting either an autosomal recessive disorder or a de novo mutation. Known genetic causes of severe hypercholesterolemia were ruled out by sequencing the responsible genes (LDLRAP, LDLR, PCSK9, APOE and APOB), and sitosterolemia was ruled out by documenting a normal plasma sitosterol:cholesterol ratio. Sequencing revealed 3 797 207 deviations from the reference sequence, of which 9726 were nonsynonymous single-nucleotide substitutions. A total of 9027 of the nonsynonymous substitutions were present in dbSNP or in 21 additional individuals from whom complete exonic sequences were available. The 699 novel nonsynonymous substitutions were distributed among 604 genes, 23 of which were single-copy genes that each contained 2 nonsynonymous substitutions consistent with an autosomal recessive model. One gene, ABCG5, had two nonsense mutations (Q16X and R446X). This finding indicated that the infant has sitosterolemia. Thus, whole-genome sequencing led to the diagnosis of a known disease with an atypical presentation. Diagnosis was confirmed by the finding of severe sitosterolemia in a blood sample obtained after the infant had been weaned. These findings demonstrate that whole-genome (or exome) sequencing can be a valuable aid to diagnose genetic diseases, even in individual patients.