Plasma apoE Levels Research Articles

Stimulation of Nitric Oxide Synthase Activity By Plasma Apolipoproteins: a Biomarker of Endothelial Function in Adults with Sickle Cell Disease

Nitric oxide (NO) plays a critical role in maintaining basal vascular tone and regulating blood flow. Many factors, including shear stress and endogenous ligands such as vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF1), stimulate the activity of endothelial nitric oxide synthase (eNOS). We have developed a bioassay that measures stimulation of NOS activity by soluble factors in unfractionated human plasma in cultured human endothelial cells using a sensitive NOS activity assay with radiolabeled substrate.The addition of 1% human plasma to the culture medium stimulates NOS activity 1.5-fold over background, with a linear response up to 10% plasma, which activates NOS 5.5-fold. We have concluded from several lines of evidence that this NOS-inducing activity in human plasma comes from high-density lipoprotein (HDL): The activity is heat-labile and sensitive to reducing agents; it is precipitable by ammonium sulfate and elutes as a broad peak on molecular exclusion and anion exchange columns; by immunoblot, the active fractions have large amounts of apolipoproteins apoA-I, apoE and paroxonase-1. We find that plasma from an apoA-I null mouse shows less stimulation of NOS activity in our bioassay than plasma from a wild type mouse and apoA-I transgenic mouse (p=0.06, Brown-Forsythe test; p=0.04, post-test for linear trend; see figure). Purified HDL fractions stimulate NOS activity four-fold, equivalent to unfractionated plasma, but further subfractionation of HDL components extinguishes its ability to activate NOS.Remarkably, higher than median NOS-stimulating activity in our bioassay was associated with endothelial-dependent blood flow, detected by venous occlusion strain gauge plethysmography measurement of forearm blood flow induced by graded infusions of acetylcholine into the brachial artery in adults with sickle cell anemia (p=0.0013, two-way ANOVA, see figure). The NOS-stimulating activity in patient plasma also correlated with the plasma apoA-I level (Spearman r=0.64, p=0.0012).In summary, our results suggest that circulating functional apoA-I and possibly other apolipoproteins, regulate NO production and endothelial function in adults with sickle cell disease, which is consistent with previous lines of evidence published by other investigators in subjects without sickle cell disease. Most strikingly, our plasma NOS stimulation assay appears to provide a useful research biomarker for endothelial function, applicable to frozen archived plasma biospecimens. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human Apolipoprotein a-I Decreases Platelet Activation Responses and Protects Mice from Arterial Thrombosis

Introduction: High-density lipoprotein (HDL) protects against thromboembolic coronary disease via reverse cholesterol transport mediated by HDL’s primary lipoprotein subunit, apolipoprotein A-I (apoA-I), binding to scavenger receptor BI (SR-BI). Absence of SR-BI increases platelet aggregation and venous/arterial thrombosis in mice, consistent with an antithrombotic role of this signaling axis. To date, the effects of the isolated apoA-I subunit on platelet activation remain unknown. We hypothesize that the antithrombotic effect is mediated by apoA-I signaling through platelet-specific SR-BI.Methods: Platelet function assays were performed on samples from healthy human volunteers (n=3). ApoA-I levels were determined using an ELISA kit. Microfluidic analysis of platelet aggregate formation on collagen under physiologic flow conditions (650 sec-1) was carried out in whole blood samples. Light transmission platelet aggregation in response to collagen and ristocetin was conducted using both platelet rich plasma (PRP) and washed platelets (WP). Venous and arterial thromboses were induced in wild type C57Bl/6 mice in a collagen (0.3 mg/kg)/epinephrine (0.03 mg/kg)-induced pulmonary embolism model and a 6% ferric chloride (FeCl3)-induced carotid artery thrombosis model, respectively. Recombinant human apoA-I was used with a final concentration of 300 µg/mL.Results/Discussion: ApoA-I plasma levels in the subjects studied fell within previously published ranges. Microfluidic analysis of platelet aggregate formation on collagen under shear stress did not demonstrate significant difference in total surface area coverage (n=3 in quadruplicate), but apoA1-treated samples demonstrated a significantly greater proportion of small aggregates (3-10 platelets/aggregate, 40.2% vs 31.8%, p<0.0001), compared to controls which had a significantly greater proportion of large aggregates (51-100 plts/agg, 6.3 vs 2.3%, p<0.001 and >100 plts/agg, 2.3% vs 0.7%, p<0.05). Platelet aggregation studies (Fig. 1) revealed significantly decreased collagen-induced aggregation in platelets treated with apoA-I compared to vehicle-treated controls in both PRP and WP samples, suggesting direct action of apoA-I on platelets. The difference was less pronounced in ristocetin-induced aggregation, though still significant, consistent with decreased activation of VWF, which has been recently shown to bind apoA-I. Apoa-I-treated PRP samples also had significantly decreased dense granule (ATP) release compared to non-treated samples. Pre-treatment with native HDL had no significant effect, consistent with studies demonstrating that only oxidized HDL inhibits aggregation. Recombinant human SR-BI (rhSR-BI) alone had little effect in PRP but inhibited collagen-induced aggregation in WP. Addition of rhSR-BI and apoA-I together inhibited aggregation in both PRP and WP to a greater degree than either component alone. Rabbit anti-human SR-BI alone had little effect in PRP but inhibited collagen-induced aggregation in WP. FeCl3 applicationinduced initial arterial occlusion within 8.8 +/-0.8 minutes in apoA-I-treated mice treated (n=3) compared to 6.1 +/- 0.3 minutes in vehicle-treated controls (n=3, p<0.01, t-test). Systemic thrombosis resulting in pulmonary embolism was induced by injection of collagen/epinephrine. Median time of survival after injection in mice pre-treated with apoA-I (n=5) was 30 +/- 0 minutes, compared to 3.3 +/- 0.4 minutes in vehicle-treated controls (n=5, p<0.001, t-test). Experiments were stopped after 30 minutes, at which time all apoA-1-treated mice were still alive, compared to zero controls.Conclusion: Though the mechanism is not yet completely understood, the data show an inhibitory effect of isolated human apoA-I on human platelet activation and murine arterial/venous thrombosis, likely through platelet inhibition. The increased inhibition seen with rhSR-BI and apoA-I together is consistent with previous studies demonstrating SR-BI’s regulatory role in platelet function. Ongoing studies include increasing sample size for human platelet function assays and murine venous/arterial thrombosis models in wild type mice with and without infusion of apoA-I compared to SR-BI-/- and apoA-I-/- mice with and without infusion of apoA-I. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Defective functionality of HDL particles in familial apoA-I deficiency: relevance of alterations in HDL lipidome and proteome

To evaluate functional and compositional properties of HDL in subjects from a kindred of genetic apoA-I deficiency, two homozygotes and six heterozygotes, with a nonsense mutation at APOA1 codon -2, Q[-2]X, were recruited together with age- and sex-matched healthy controls (n = 11). Homozygotes displayed undetectable plasma levels of apoA-I and reduced levels of HDL-cholesterol (HDL-C) and apoC-III (5.4% and 42.6% of controls, respectively). Heterozygotes displayed low HDL-C (21 ± 9 mg/dl), low apoA-I (79 ± 24 mg/dl), normal LDL-cholesterol (132 ± 25 mg/dl), and elevated TG (130 ± 45 mg/dl) levels. Cholesterol efflux capacity of ultracentrifugally isolated HDL subpopulations was reduced (up to -25%, P < 0.01, on a glycerophospholipid [GP] basis) in heterozygotes versus controls. Small, dense HDL3 and total HDL from heterozygotes exhibited diminished antioxidative activity (up to -48%, P < 0.001 on a total mass basis) versus controls. HDL subpopulations from both homozygotes and heterozygotes displayed altered chemical composition, with depletion in apoA-I, GP, and cholesteryl ester; enrichment in apoA-II, free cholesterol, and TG; and altered phosphosphingolipidome. The defective atheroprotective activities of HDL were correlated with altered lipid and apo composition. These data reveal that atheroprotective activities of HDL particles are impaired in homozygous and heterozygous apoA-I deficiency and are intimately related to marked alterations in protein and lipid composition.

Abstract 15426: CSL112 Enhances Cholesterol Efflux Equally in Patients with High and Low High Density Lipoprotein Functionality

Introduction: The ability of high-density lipoprotein (HDL) to remove cholesterol from macrophages in atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. CSL112 is a novel formulation of apoA-I purified from human plasma and reconstituted to form HDL particles suitable for infusion. Two phase 1 studies in healthy subjects and one phase 2a study in coronary heart disease (CHD) patients have demonstrated favorable safety, pharmacokinetics and biomarker responses to single or multiple infusions of CSL112. The aim of the current study was to assess the influence of subject-specific HDL functionality, sometimes described as HDL dysfunction, on the pharmacodynamic effects of CSL112. Methods and Results: To assess HDL functionality, we measured the specific cholesterol efflux activity of apoA-I (cholesterol efflux divided by apoA-I) in subjects at baseline. CHD patients (mean ± SD = 7.3 ± 0.3 %efflux per g/L apoA-I (n=43)) had lower activity (p&lt;0.001) than healthy subjects (mean ± SD = 8.8 ± 0.2 % efflux per g/L apoA-I (n=93)), consistent with prior work showing impaired HDL function in CHD patients. Every subject infused with CSL112 responded, showing increased apoA-I plasma levels and serum cholesterol efflux. To assess changes in HDL functionality upon infusion of CSL112, we plotted changes in cholesterol efflux vs changes in apoA-I. To account for variations of these analytes over time and dose, we plotted area under curve (AUC) for 0 to 24 h after infusion of various doses of CSL112. In all three studies, AUC for apoA-I correlated with the AUC for cholesterol efflux, with coincident lines, indicating equal elevation in efflux among healthy and CHD patients. To further examine this relationship, subjects were stratified into tertiles based on specific activity. Infusion of CSL112 elevated efflux similarly in subjects in the highest HDL functionality and lowest HDL functionality tertiles. Conclusion: ApoA-I from CHD patients compared to healthy subjects had lower specific activity in regards to cholesterol efflux. CSL112 causes strong elevation in cholesterol efflux regardless of HDL function and represents a promising candidate to rapidly remove plaque cholesterol and reduce recurrent atherothrombotic events.

Lower plasma apolipoprotein A1 levels are found in Parkinson's disease and associate with apolipoprotein A1 genotype.

The discovery of novel plasma-based biomarkers could lead to new approaches in the treatment of Parkinson's disease (PD). Here, we explore the role of plasma apolipoprotein A1 (ApoA1) as a risk marker for PD and evaluate the influence of APOA1 promoter variation on plasma ApoA1 levels. Plasma ApoA1 and the single-nucleotide polymorphism, rs670, were assayed in a discovery cohort (cohort 1) of 301 PD patients, 80 normal controls (NCs), and 165 subjects with other neurodegenerative diseases, as well as a cohort (cohort 2) of 158 PD patients from a second clinical site. Additionally, rs670 was genotyped in a third cohort of 1,494 PD and 925 NC subjects from both clinical sites. Compared to both normal and disease controls, PD patients have lower plasma ApoA1 (P < 0.001 for both comparisons). Moreover, in PD patients, plasma ApoA1 levels are correlated with genotype at the APOA1 promoter polymorphism, rs670. Specifically, lower plasma ApoA1 levels were found in rs670 major allele (G) homozygotes in both cohort 1 (P = 0.009) and in a replication cohort (cohort 2; n = 158 PD patients; P = 0.024). Finally, evaluating rs670 genotype frequencies in 1,930 PD cases versus 997 NCs, the rs670 GG genotype shows a trend toward association (odds ratio: 1.1; P = 0.10) with PD. Our results are compatible with a model whereby circulating ApoA1 levels may be useful in risk-stratifying subjects for the development of PD, with higher ApoA1 levels suggesting relative protection. Future studies evaluating modulation of ApoA1 as a novel therapeutic strategy in PD are warranted. © 2014 International Parkinson and Movement Disorder Society.

ApoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.

A novel ApoA-I truncation (ApoA-IMytilene) associated with decreased ApoA-I production

ObjectiveWe report a novel apolipoprotein (apo) A-I truncation (apoA-IMytilene) due to a heterozygous nonsense mutation (c.718C > T, p.Gln216*) in a 68-year-old male proband with premature coronary heart disease (CHD), corneal arcus, and very low plasma concentrations of HDL cholesterol (HDL-C) and apoA-I. Two family members also had the same mutation. Our objectives were to characterize the kindred and to examine the kinetics of apoA-I, as well as cellular cholesterol efflux capacity in the proband. MethodsWe carried out the kinetic studies using a primed constant infusion of [5,5,5-D3]L-leucine and isotopic enrichment was determined by gas chromatography mass spectrometry in the proband and seven controls with low HDL-C. To assess cellular cholesterol efflux capacity, we used a validated ex vivo system that involved incubation of J774 macrophages with apoB-depleted serum from the proband, five controls with normal HDL-C, and two controls with low HDL-C. ResultsStable isotope kinetic studies indicated that the proband had an apoA-I production rate (PR) that was 41% lower than the mean PR observed in low HDL-C controls (n = 7). The cellular cholesterol efflux capacity assessment showed normalized cholesterol efflux capacity in the proband was decreased by 36% compared to the mean normalized cholesterol efflux capacity of normal controls (n = 5). ConclusionsOur data indicate that this novel heterozygous apoA-I truncation is associated with markedly decreased levels of HDL-C, plasma apoA-I, and apoA-I in large α-1 HDL particles, as well as decreased total cellular cholesterol efflux and decreased apoA-I production.

Abstract 584: ApoE3[K146N/R147W] Acts as a Dominant Negative ApoE Form that Prevents Remnant Clearance and Inhibits the Biogenesis of HDL

Introduction: The K146N/R147W substitutions in human apolipoproteinE3 (apoE3) have been associated with a dominant form of type III hyperlipoproteinemia which is expressed at an early age. Methods: The effects of the K146N,R147W substitutions on the lipid and lipoprotein profiles and the HDL phenotypes were studied by adenovirus mediated gene transfer of the full length and a truncated apoE3[K146N/R147W]-202 mutant using different mouse models. Results: A low dose of adenovirus expressing the apoE3[K146N/R147W] mutant in apoE deficient or in apoA-I x apoE double deficient mice exacerbated the hypercholesterolemia and increased greatly plasma apoE and triglycerides levels. In apoE deficient mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and resulted in the accumulation of discoidal apoE containing HDL particles in plasma. Similar doses of WT apoE3 cleared the cholesterol of apoE deficient mice without induction of hypertriglyceridemia and promoted formation of spherical HDL particles. A unique feature of the truncated apoE3[K146N/R147W]-202 mutant is that it prevented the induction of hypertriglyceridemia but did not correct the hypercholesterolemia. Other apoE-202 truncated mutants tested did not induce hypertriglyceridemia but corrected hypercholesterolemia. Infection of apoE deficient mice with adenoviruses expressing the apoE3[K146N/R147W] and lipoprotein lipase corrected the hypertriglyceridemia but did not prevent the formation of discoidal HDL particles. Infection of apoE deficient mice with adenoviruses expressing the apoE3[K146N/R147W] and lecithin:cholesterol (LCAT) acyltransferase corrected hypertriglyceridemia, normalized the plasma cholesteryl ester to total cholesterol ratio and generated spherical HDL particles. The combined data indicate that the K146N/R147W substitutions in the full length and the truncated apoE3[K146N/R147W] mutant prevent receptor mediated remnant clearance. Conclusion: The lipid/lipoprotein and HDL abnormalities observed in the different mouse models suggest that the apoE3[K146N/R147W] mutant acts a dominant negative ligand that exacerbates the dyslipidemia and also affects the activity of LCAT and thus inhibits the maturation of HDL.

A lincRNA-DYNLRB2-2/GPR119/GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis

Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macrophages and atherosclerotic plaque progression in apoE(-/-) mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. In vivo, apoE(-/-) mice were randomly divided into two groups and infected with lentivirus (LV)-Mock or LV-GPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1β, IL-6, and TNF-α levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibited by infection of apoE(-/-) mice with LV-GPR119. Our findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atherosclerosis in apoE(-/-) mice. These suggest that GPR119 may be a promising candidate as a therapeutic agent.

Total apolipoprotein E levels and specific isoform composition in cerebrospinal fluid and plasma from Alzheimer’s disease patients and controls

The apolipoprotein E (ApoE) ε4 allele is the strongest risk factor of sporadic Alzheimer's disease (AD), however, the fluid concentrations of ApoE and its different isoforms (ApoE2, ApoE3 and ApoE4) in AD patients and among APOE genotypes (APOE ε2, ε3, ε4) remain controversial. Using a novel mass spectrometry-based method, we quantified total ApoE and specific ApoE isoform concentrations and potential associations with age, cognitive status, cholesterol levels and established AD biomarkers in cerebrospinal fluid (CSF) from AD patients versus non-AD individuals with different APOE genotypes. We also investigated plasma total ApoE and ApoE isoform composition in a subset of these individuals. In total n = 43 AD and n = 43 non-AD subjects were included. We found that CSF and plasma total ApoE levels did not correlate with age or cognitive status and did not differ between AD and non-AD subjects deeming ApoE as an unfit diagnostic marker for AD. Also, whereas CSF ApoE levels did not vary between APOE genotypes APOE ε4 carriers exhibited significantly decreased plasma ApoE levels attributed to a specific decrease in the ApoE4 isoform concentrations. CSF total ApoE concentrations were positively associated with CSF, total tau, tau phosphorylated at Thr181 and Aβ1-42 of which the latter association was weaker and only present in APOE ε4 carriers indicating a differential involvement of ApoE in tau versus Aβ-linked neuropathological processes. Future studies need to elucidate whether the observed plasma ApoE4 deficiency is a life-long condition in APOE ɛ4 carriers and whether this decrease in plasma ApoE predisposes APOE ɛ4 carriers to AD.

Short term resistance training enhanced plasma apoA-I and FABP4 levels in Streptozotocin-induced diabetic rats

BackgroundType 1 diabetes mellitus is associated with a high risk for early atherosclerotic complications. Altered lipids and lipoprotein metabolism in chronic diabetes mellitus is associated with pathogenesis of atherosclerosis and other cardiovascular diseases. The aim of this study was to investigate the effects of 4 weeks resistance training on plasma lipid profile, fatty acid binding protein (FABP) 4 and apolipoprotein (apo) A-I levels in type 1 diabetic rats.MethodsThirty two male Wister rats (12–14 weeks old) were randomly divided into four groups: non-diabetic control; non-diabetic trained; diabetic control; diabetic trained. The rats in training groups were subjected to a resistance training program (3 days/wk, for 4 wk) consisted of climbing a ladder carrying a load suspended from the tail.ResultsDiabetic inducing increased plasma apoA-I and decreased FABP4 levels compared with non-diabetic control group (respectively, P = 0.001 & P = 0.041). After 4 weeks’ resistance training, plasma levels of apoA-I and FABP4 in the diabetic trained rats were significantly higher compared with the diabetic control group (respectively, P = 0.003 & P = 0.017). Plasma HDL-C level in diabetic trained group was higher than diabetic control group (P = 0.048). Liver triglycerides concentrations were significantly lower in both trained (non-diabetic and diabetic) groups compared with their control groups (respectively, P = 0.041 and P = 0.002).ConclusionThese data indicated that resistance training may be an efficient intervention strategy to increase plasma apoA-I, HDL-C and FABP4 concentrations, along with decreases liver triglycerides in streptozotocin induced diabetic rats. Further research is needed to elucidate physiological significance of circulating FABP4 levels.

Meta-Analysis of Peripheral Blood Apolipoprotein E Levels in Alzheimer’s Disease

BackgroundPeripheral blood Apolipoprotein E (ApoE) levels have been proposed as biomarkers of Alzheimer’s disease (AD), but previous studies on levels of ApoE in blood remain inconsistent. This meta-analysis was designed to re-examine the potential role of peripheral ApoE in AD diagnosis and its potential value as a candidate biomarker.MethodsWe conducted a systematic literature search of MEDLINE, EMBASE, the Cochrane library, and BIOSIS previews for case-control studies measuring ApoE levels in serum or plasma from AD subjects and healthy controls. The pooled weighted mean difference (WMD) and 95% confidence interval (CI) were used to estimate the association between ApoE levels and AD risk.ResultsEight studies with a total of 2250 controls and 1498 AD cases were identified and analyzed. The pooled WMD from a random-effect model of AD participants compared with the healthy controls was −5.59 mg/l (95% CI: [−8.12, −3.06]). The overall pattern in WMD was not varied by characteristics of study, including age, country, assay method, publication year, and sample type.ConclusionsOur meta-analysis supports a lowered level of blood ApoE in AD patients, and indicates its potential value as an important risk factor for AD. Further investigation employing standardized assay for ApoE measurement are still warranted to uncover the precise role of ApoE in the pathophysiology of AD.

Gender-specific associations of the APOA1 − 75G > A polymorphism with several metabolic syndrome components in Turkish adults

BackgroundVariations in the apolipoprotein A-1 (APOA1) gene, a determinant of plasma high-density lipoprotein cholesterol (HDL-C) and apoA-I levels, may contribute to cardiovascular diseases. We evaluated the effects of a promoter polymorphism (−75G>A) in the APOA1 gene on metabolic syndrome (MetS) components in a Turkish population sample. MethodsRandomly selected 1515 Turkish adults (age 49.9±11.8years, 785 females) were genotyped for −75G>A polymorphism using hybridization probes in Real-Time PCR LC480 device. MetS and atherogenic dyslipidemia were defined using the criteria of ATP III. ResultsThe −75AA genotype prevailed in 3.9% of men and 2.4% of women, and was independently associated with significantly higher HDL-C concentrations. Independent associations with the −75GA genotype existed only in men: higher diastolic and systolic blood pressure (BP) levels (p<0.05) were observed in male −75GA heterozygotes. Logistic regression revealed that the GA genotype confers elevated risk for atherogenic dyslipidemia (OR=1.57, 95% Cl 1.06–2.3) after adjustment for associated risk factors. Independent associations with atherogenic dyslipidemia or elevated BP did not emerge in women. ConclusionAPOA1 −75G>A polymorphism is independently related to HDL-C concentrations. Independent associations of the −75GA genotype with elevated BP and atherogenic dyslipidemia were confined to men. These gender-modulated associations suggest novel gene–gender–environmental interactions.

Both Targeted Mass Spectrometry and Flow Sorting Analysis Methods Detected the Decreased Serum Apolipoprotein E Level in Alzheimer's Disease Patients

Apolipoprotein E (ApoE) polymorphism has been appreciated as a valuable predictor of Alzheimer disease (AD), and the associated ε4 allele has been recognized as an indicator of susceptibility to this disease. However, serum ApoE levels have been a controversial issue in AD, due to the great variability regarding the different target detection methods, ethnicity, and the geographic variations of cohorts. The aim of this study was to validate serum ApoE levels in relation to AD, particularly using two distinct detection methods, liquid chromatography-selected reaction monitoring (SRM) mass spectrometry and microsphere-based fluorescence-activated cell sorting (FACS) analysis, to overcome experimental variations. Also, comparison of serum ApoE levels was performed between the level of protein detection by FACS and peptide level by SRM in both control and AD patients. Results from the two detection methods were cross-confirmed and validated. Both methods produced fairly consistent results, showing a significant decrease of serum ApoE levels in AD patients relative to those of a control cohort (43 control versus 45 AD, p < 0.0001). Significant correlation has been revealed between results from FACS and SRM (p < 0.0001) even though lower serum ApoE concentration values were measured in protein by FACS analysis than in peptide-level detections by SRM. Correlation study suggested that a decrease of the serum ApoE level in AD is related to the mini-mental state exam score in both results from different experimental methods, but it failed to show consistent correlation with age, gender, or clinical dementia rating.

Human Apolipoprotein E Resequencing by Proteomic Analysis and Its Application to Serotyping

BackgroundApolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer’s disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.MethodsCombining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.ResultsThe mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.ConclusionOur novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.

Apolipoprotein E–low density lipoprotein receptor interaction affects spatial memory retention and brain ApoE levels in an isoform-dependent manner

Human apolipoprotein E (apoE) exists in three isoforms: apoE2, apoE3 and apoE4. APOE ε4 is a major genetic risk factor for cardiovascular disease (CVD) and Alzheimer's disease (AD). ApoE mediates cholesterol metabolism by binding various receptors. The low-density lipoprotein receptor (LDLR) has a high affinity for apoE, and is the only member of its receptor family to demonstrate an apoE isoform specific binding affinity (E4>E3>>E2). Evidence suggests that a functional interaction between apoE and LDLR influences the risk of CVD and AD. We hypothesize that the differential cognitive effects of the apoE isoforms are a direct result of their varying interactions with LDLR. To test this hypothesis, we have employed transgenic mice that express human apoE2, apoE3, or apoE4, and either human LDLR (hLDLR) or no LDLR (LDLR−/−). Our results show that plasma and brain apoE levels, cortical cholesterol, and spatial memory are all regulated by isoform-dependent interactions between apoE and LDLR. Conversely, both anxiety-like behavior and cued associative memory are strongly influenced by APOE genotype, but these processes appear to occur via an LDLR-independent mechanism. Both the lack of LDLR and the interaction between E4 and the LDLR were associated with significant impairments in the retention of long term spatial memory. Finally, levels of hippocampal apoE correlate with long term spatial memory retention in mice with human LDLR. In summary, we demonstrate that the apoE-LDLR interaction affects regional brain apoE levels, brain cholesterol, and cognitive function in an apoE isoform-dependent manner.

CSF Apo-E levels associate with cognitive decline and MRI changes

Apolipoprotein E (APOE) ε4 allele is the most important genetic risk factor for Alzheimer's disease (AD) and it is thought to do so by modulating levels of its product, apolipoprotein E (Apo-E), and regulating amyloid-β (Aβ) clearance. However, information on clinical and biomarker correlates of Apo-E proteins is scarce. We examined the relationship of cerebrospinal fluid (CSF) and plasma Apo-E protein levels, and APOE genotype to cognition and AD biomarker changes in 311 AD neuroimaging initiative subjects with CSF Apo-E measurements and 565 subjects with plasma Apo-E measurements. At baseline, higher CSF Apo-E levels were associated with higher total and phosphorylated CSF tau levels. CSF Apo-E levels were associated with longitudinal cognitive decline, MCI conversion to dementia, and gray matter atrophy rate in total tau/Aβ1-42 ratio and APOE genotype-adjusted analyses. In analyses stratified by APOE genotype, our results were only significant in the group without the ε4 allele. Baseline CSF Apo-E levels did not predict longitudinal CSF Aβ or tau changes. Plasma Apo-E levels show a mild correlation with CSF Apo-E levels, but were not associated with longitudinal cognitive and MRI changes. Based on our analyses, we speculate that increased CSF Apo-E2 or -E3 levels might represent a protective response to injury in AD and may have neuroprotective effects by decreasing neuronal damage independent of tau and amyloid deposition in addition to its effects on amyloid clearance.

Apolipoprotein E Gene Variants on the Risk of End Stage Renal Disease

ObjectiveEnd-stage renal disease (ESRD) is a severe health concern over the world. Associations between apolipoprotein E (apoE) gene polymorphisms and the risk of ESRD remained inconclusive. This study aimed to investigate the association between apoE gene polymorphisms and ESRD susceptibility.MethodsDatabases including PubMed, Embase, Web of Science and the Cochrane Library were searched to find relevant studies. Meta-analysis method was used synthesize the eligible studies. ResultsSixteen pertinent case-control studies which included 3510 cases and 13924 controls were analyzed. A significant association was found between ε2 allele and the ESRD risk (odds ratio (OR) = 1.30, 95% confidence interval (CI) 1.15–1.46, P < 0.0001; I 2 = 18%, P for heterogeneity = 0.24). The ε2ε3, ε2ε4, ε3ε3, ε3ε4, ε4ε4, ε3 and ε4 were not associated with the susceptibility of ESRD. In the subgroup analysis by ethnicity, there was a statistically significant association between ε2ε3 or ε2 allele and ESRD risk in East Asians (OR = 1.66, 95% CI 1.31–2.10, P < 0.0001; OR = 1.62, 95% CI 1.31–2.01, P < 0.0001, respectively), but not in Caucasians. E2 carriers had higher plasma apoE (mean difference = 16.24 mg/L, 95% CI 7.76-24.73, P = 0.0002) than the (ε3 + ε4) carriers in patients with ESRD. The publication bias was not significant.ConclusionThe ε2 allele of apoE gene might increase the risk of ESRD. E2 carriers expressed higher level of plasma apoE in patients with ESRD. More well-designed studies are needed to confirm these associations in the future.

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225-230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I⁻/⁻ mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I⁻/⁻ × apoE⁻/⁻ mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225-230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225-230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218-226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I⁻/⁻ mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I⁻/⁻ × apoE⁻/⁻ mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I⁻/⁻ mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218-222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218-222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.