Total Apo Research Articles

Abstract 4140115: Comparison of Lipoprotein(a) and Other Apo B Containing Lipoproteins as Predictors of Major Adverse Cardiovascular Events in ODYSSEY OUTCOMES

Background: Lipoprotein(a) [Lp(a)] and other atherogenic lipoproteins each contain one molecule of apolipoprotein B (apoB) per particle. Recent studies have suggested that on a per particle basis, Lp(a) is more strongly associated with major adverse cardiovascular events (MACE) than low density lipoprotein. Hypothesis: In statin-treated patients with recent acute coronary syndrome (ACS), we tested the hypothesis that, on a per particle basis, Lp(a) and its change on treatment with alirocumab (ALI) are more strongly associated with MACE than other [non-Lp(a)] apoB-containing particles. Methods: Molar apolipoprotein(a) [corresponding to Lp(a)] and apo B were measured by mass spectrometry at baseline and month 4 (M4) in a subgroup of 11,957 patients who provided consent for use of stored samples in the ODYSSEY OUTCOMES trial. Non-Lp(a) apoB in nmol/L was calculated as total apo B – Lp(a). Lp(a) and non-Lp(a) apo B at baseline in the placebo group, and absolute changes in their levels on ALI, were related to risk of MACE using proportional hazards models. The latter analysis was adjusted for baseline Lp(a) and non-Lp(a) apo B and stratified by baseline Lp(a) (<125 nmol/L and ≥125 nmol/L). Results: Baseline levels of Lp(a) and non-Lp(a) apoB are shown in the Table . In the placebo group, both baseline Lp(a) and non-Lp(a) apo B independently predicted MACE. At M4 in the ALI group, median Lp(a) change from baseline was -40.9 and -7.0 in those with baseline levels ≥ or <125 nmol/L, respectively. Corresponding median changes in non-Lp(a) apoB were -739 and -776 nmol/L (all P<0.001). In the ALI group with baseline Lp(a)>125 nmol/L, the decrease from baseline in Lp(a), but not the decrease in non-Lp(a) apo B, was significantly related to risk of MACE. Among those with baseline Lp(a)≤125 nmol/L, the decrease from baseline in non-Lp(a) apo B, but not in Lp(a), with ALI was significantly related to risk of MACE. Conclusions: On a per particle basis, baseline Lp(a) and non-Lp(a) apo B both predicted MACE. Among those with high baseline Lp(a), reduction in MACE with ALI was predominantly associated with reduction of Lp(a); among those with lower baseline Lp(a) reduction in MACE with ALI was predominantly associated with reduction in non-Lp(a) apo-B. Lp(a) may be an important target of treatment with ALI in those with elevated levels after ACS.

Effects of the subtypes of apolipoprotein E on immune inhibition and prognosis in patients with Hepatocellular Carcinoma

PurposeTo investigate whether prognosis of patients with hepatocellular carcinoma (HCC) is affected by the abundance and subgroups of myeloid-derived suppressor cells (MDSCs) as well as subtypes and expression of apolipoprotein E (apoE).Methods31 HCC patients were divided into three groups according to blood total apoE level for detecting the abundance of immunoregulatory cells by flow cytometry. Tumour tissue microarrays from 360 HCC patients were evaluated about the abundance and subgroups of MDSCs and the expression of apoE2, apoE3, apoE4 by immunofluorescence staining and immunohistochemistry staining. Survival analysis by means of univariate, multivariate COX regression and Kaplan-Meier methods of the 360 patients was performed based on clinical and pathological examinations along with 10 years’ follow-up data.ResultsThe lower apoE group presented higher abundance of MDSCs in the peripheral blood of HCC patients than higher apoE group. The abundance of monocyte-like MDSCs (M-MDSCs) was higher in the apoE low level group than high level group (p = 0.0399). Lower H-score of apoE2 (HR = 6.140, p = 0.00005) and higher H-score of apoE4 (HR = 7.001, p = 0.009) in tumour tissue were significantly associated with shorter overall survival (OS). The higher infiltration of polymorphonuclear granulocyte-like MDSCs (PMN-MDSCs, HR = 3.762, p = 0.000009) and smaller proportion of M-MDSCs of total cells (HR = 0.454, p = 0.006) in tumour tissue were independent risk factors for shorter recurrence-free survival (RFS).ConclusionThe abundance of MDSCs in HCC patients’ plasma negatively correlates with the level of apoE. The expression of apoE4 in HCC tissue indicated a poor prognosis while apoE2 might be a potential protective factor.

Plasma apolipoprotein E levels, isoform composition, and dimer profile in relation to plasma lipids in racially diverse patients with Alzheimer’s disease and mild cognitive impairment

BackgroundThe APOEε4-promoted risk of Alzheimer’s disease (AD) is lower in Black/African-Americans (B/AAs), compared to non-Hispanic whites (NHWs). Previous studies reported lower plasma apolipoprotein E (apoE) levels in NHW APOEε4-carriers compared to non-carriers, and low plasma apoE levels were directly associated with an increased risk of AD and all dementia. We further showed that APOEε3/ε3 AD patients exhibited reduced plasma apoE dimers compared to corresponding control subjects. Whether plasma apoE levels and apoE dimer formation differ between races/ethnicities and therefore may help explain AD risk racial disparity remains to be elucidated.MethodsUsing mass spectrometry, we determined total plasma apoE and apoE isoform levels in a cohort of B/AAs (n = 58) and NHWs (n = 67) including subjects with normal cognition (B/AA: n = 25, NHW: n = 28), mild cognitive impairment (MCI) (B/AA: n = 24, NHW: n = 24), or AD dementia (B/AA: n = 9, NHW: n = 15). Additionally, we used non-reducing western blot analysis to assess the distribution of plasma apoE into monomers/disulfide-linked dimers. Plasma total apoE, apoE isoform levels, and % apoE monomers/dimers were assessed for correlations with cognition, cerebrospinal fluid (CSF) AD biomarkers, sTREM2, neurofilament light protein (NfL), and plasma lipids.ResultsPlasma apoE was predominantly monomeric in both racial groups and the monomer/dimer distribution was not affected by disease status, or correlated with CSF AD biomarkers, but associated with plasma lipids. Plasma total apoE levels were not related to disease status and only in the NHW subjects we observed lower plasma apoE levels in the APOEε4/ε4-carriers. Total plasma apoE levels were 13% higher in B/AA compared to NHW APOEε4/ε4 subjects and associated with plasma high-density lipoprotein (HDL) in NHW subjects but with low-density lipoprotein levels (LDL) in the B/AA subjects. Higher plasma apoE4 levels, exclusively in APOEε3/ε4 B/AA subjects, were linked to higher plasma total cholesterol and LDL levels. In the controls, NHWs and B/AAs exhibited opposite associations between plasma apoE and CSF t-tau.ConclusionsThe previously reported lower APOEε4-promoted risk of AD in B/AA subjects may be associated with differences in plasma apoE levels and lipoprotein association. Whether differences in plasma apoE levels between races/ethnicities result from altered APOEε4 expression or turnover, needs further elucidation.

156-OR: Apolipoprotein C-II Posttranslational Truncation Is Associated with Reduced Incident Cardiovascular Risk in Multiethnic Study of Atherosclerosis (MESA)

Higher truncated-to-native proteoform ratios of apolipoproteins (apo) C-I (C-I’/C-I) and C-II (C-II’/C-II) are associated with lower triglycerides (TG) and higher HDL levels. Here we examined the relationships of C-I’/C-II and C-II’/C-II, and total apo C-I and C-II concentrations with atherosclerotic cardiovascular disease (ASCVD) and coronary heart disease (CHD). Apo C-I and C-II proteoforms were measured by mass spectrometry immunoassay at baseline in 5,765 MESA participants followed for CHD (definite myocardial infarction, resuscitated cardiac arrest or fatal CHD) and ASCVD (CHD or stroke) for up to 17 years. Baseline total apo C-I and C-II levels were measured in a subgroup of 3,848 participants. After adjusting for age, gender, and race/ethnicity, lower C-II’/C-II was associated with both ASCVD and CHD (Figure). The association of C-II’/C-II with CHD remained significant after further adjusting for BMI, diabetes, systolic BP, eGFR, use of tobacco, statins, antihypertensive medications, total apoC-II, and plasma TG, but was abolished after adjusting for HDL. Neither ASCVD nor CHD were related to total apo C-I and C-II or CI’/CI. In conclusion, increased posttranslational apoC-II truncation (but not total apo C-I or C-II) is associated with reduced cardiovascular risk, possibly explained by differences in HDL cholesterol metabolism. Disclosure J.Koska: None. J.Furtado: Employee; Biogen. Y.Hu: None. D.Nedelkov: None. D.Billheimer: Advisory Panel; AstraZeneca. M.Budoff: Research Support; Novo Nordisk, Novartis, Boehringer Ingelheim Inc., Speaker's Bureau; Boehringer Ingelheim and Eli Lilly Alliance. M.Allison: None. R.Mcclelland: None. P.Reaven: Research Support; Dexcom, Inc. Funding National Heart, Lung, and Blood Institute (R01HL138969, 75N92020D00001, HHSN268201500003I, N01HC95159, 75N92020D00005, N01HC95160, 75N92020D00002, N01HC95161, 75N92020D00003, N01HC95162, 75N92020D00006, N01HC95163, 75N92020D00004, N01HC95164, 75N92020D00007, N01HC95165, N01HC95166, N01HC95167, N01HC95168, N01HC95169); National Center for Advancing Translational Sciences (UL1TR000040, UL1TR001079, UL1TR001420)

449-P: Truncations of Apolipoproteins C-I and C-II Are Associated with Coronary Artery Calcium Progression in Multiethnic Study of Atherosclerosis (MESA)

Higher truncated-to-native proteoform ratios of apolipoproteins (apo) C-I (C-I’/C-I) and C-II (C-II’/C-II) are associated with less atherogenic lipid profiles, including lower triglycerides (TG) and higher HDL levels. Here we examined the relationships of C-I’/C-II and C-II’/C-II, and total apoC-I and apoC-II concentrations with coronary artery calcium (CAC) progression. Apo C-I and C-II proteoforms were measured by mass spectrometry immunoassay in 5,791 baseline MESA plasma samples. Total apo C-I and C-II concentrations were assayed in 3,851 samples. CAC was measured 1 to 4 times (mean 2.5) over 10 years. Repeated measures regression models were adjusted for age, gender, race/ethnicity, and follow-up time (Model 1), then for BMI, diabetes, systolic BP, use of tobacco, statins and antihypertensive medications (Model 2), and then for plasma HDL and TG (Model 3). Among 5,069 participants with at least one follow-up CAC CT scan, 2,513 had CAC progression defined as events of CAC changing from 0 to > 0, or ≥ 10 units/yr from CAC > 0 but ≤ 100, or ≥ 10%/yr from CAC > 100. Progression was inversely associated with C-I’/C-I (Risk Ratio per 1 SD: 0.93 [95% CI 0.87, 0.97], Model 3). In those with baseline CAC = 0 (n = 2,581), risk of CAC > 0 (897 events) was inversely associated with C-I’/C-I in Model 1 only (0.85 [0.79, 0.93]). In those with baseline CAC > 0, C-II’/C-II was positively associated with follow-up change in density score (β-estimate per 1 SD 0.02 [95% CI: 0.001, 0.03] units) in Model 3, indicating a potentially more stable plaque. Baseline-adjusted follow-up density score was also positively related to total apoC-I in Model 2 (0.02 [0.004, 0.04]). None of the CAC measures were related to total apoC-II. In conclusion, increased truncation of apo C-I and C-II is associated with reduced CAC progression and/or improved stability of coronary atherosclerotic plaque that is not explained by total concentrations of both apolipoproteins. Disclosure J.Koska: None. J.Furtado: Employee; Biogen. Y.Hu: None. D.Billheimer: Advisory Panel; AstraZeneca. M.Allison: None. M.Budoff: Research Support; Novo Nordisk, Novartis, Boehringer Ingelheim Inc., Speaker's Bureau; Boehringer Ingelheim and Eli Lilly Alliance. D.Nedelkov: None. R.Mcclelland: None. P.Reaven: Research Support; Dexcom, Inc. Funding National Heart, Lung, and Blood Institute (R01HL138969, 75N92020D00001, HHSN268201500003I, N01HC95159, 75N92020D00005, N01HC95160, 75N92020D00002, N01HC95161, 75N92020D00003, N01HC95162, 75N92020D00006, N01HC95163, 75N92020D00004, N01HC95164, 75N92020D000

Apo CIII Proteoforms, Plasma Lipids, and Cardiovascular Risk in MESA.

Apo CIII (apolipoprotein CIII) is an important regulator of triglyceride metabolism and was associated with cardiovascular risk in several cohorts. It is present in 4 major proteoforms, a native peptide (CIII0a), and glycosylated proteoforms with zero (CIII0b), 1 (CIII1, most abundant), or 2 (CIII2) sialic acids, which may differentially modify lipoprotein metabolism. We studied the relationships of these proteoforms with plasma lipids and cardiovascular risk. Apo CIII proteoforms were measured by mass spectrometry immunoassay in baseline plasma samples of 5791 participants of Multi-Ethnic Study of Atherosclerosis, an observational community-based cohort. Standard plasma lipids were collected for up to 16 years and cardiovascular events (myocardial infarction, resuscitated cardiac arrest, or stroke) were adjudicated for up to 17 years. Apo CIII proteoform composition differed by age, sex, race and ethnicity, body mass index, and fasting glucose. Notably, CIII1 was lower in older participants, men and Black and Chinese (versus White) participants, and higher in obesity and diabetes. In contrast, CIII2 was higher in older participants, men, Black, and Chinese persons, and lower in Hispanic individuals and obesity. Higher CIII2 to CIII1 ratio (CIII2/III1) was associated with lower triglycerides and higher HDL (high-density lipoprotein) in cross-sectional and longitudinal models, independently of clinical and demographic risk factors and total apo CIII. The associations of CIII0a/III1 and CIII0b/III1 with plasma lipids were weaker and varied through cross-sectional and longitudinal analyses. Total apo CIII and CIII2/III1 were positively associated with cardiovascular disease risk (n=669 events, hazard ratios, 1.14 [95% CI, 1.04-1.25] and 1.21 [1.11-1.31], respectively); however, the associations were attenuated after adjustment for clinical and demographic characteristics (1.07 [0.98-1.16]; 1.07 [0.97-1.17]). In contrast, CIII0b/III1 was inversely associated with cardiovascular disease risk even after full adjustment including plasma lipids (0.86 [0.79-0.93]). Our data indicate differences in clinical and demographic relationships of apo CIII proteoforms, and highlight the importance of apo CIII proteoform composition in predicting future lipid patterns and cardiovascular disease risk.

Metrologically Traceable Quantification of 3 Apolipoprotein E Isoforms in Cerebrospinal Fluid.

APOE genotype is associated with Alzheimer disease. Thus, the concentration of apolipoprotein E (apoE) isoforms in cerebrospinal fluid (CSF) could be altered in dementia. However, conflicting results have been obtained in different studies. Carefully validated and standardized assays could improve the interpretation of research findings, allow their replication in other laboratories, and generalize their application. To evaluate this hypothesis, we aimed to develop, validate, and standardize a new measurement procedure using LC-MS/MS. Purified recombinant apoE protein standards (E2, E3, E4) were thoroughly characterized and used to assign the concentration of a matrix-matched calibration material that contained each apoE isoform, which ensured the metrological traceability of results. The assay of each isoform in human CSF was precise (≤11%CV) and of moderate throughput (approximately 80 samples per day). It demonstrated good linearity and parallelism for lumbar CSF, ventricular CSF, and bovine CSF. The use of an SI-traceable matrix-matched calibrator enabled precise and accurate measurements. There was no association observed between total apoE concentration and the number of Ɛ4 alleles in a cohort of 322 participants. However, the concentration of each isoform was significantly different in heterozygotes, with E4 > E3 > E2. Isoform concentrations were associated with cognitive and motor symptoms but contributed negligibly to a predictive model of cognitive impairment that included established CSF biomarkers. Our method simultaneously measures each apoE isoform in human CSF with excellent precision and accuracy. A secondary matrix-matched material has been developed and is available to other laboratories to improve interlaboratory agreement.

Pharmacologic inhibition of astrocyte ApoE release

AbstractBackgroundThe ApoE4 allele is the strongest known genetic risk factor for late onset Alzheimer’s disease. Despite this knowledge, therapeutic strategies seeking to modulate brain ApoE homeostasis remain largely unexplored. Several lines of evidence demonstrate that astrocytes function as the primary producers of ApoE within the brain. In this study, we examine how targeted disruption of astrocyte lipid metabolism and protein prenylation effect the secretion of ApoE from primary astrocyte cultures.MethodPrimary cell cultures of mouse astrocytes were grown from 1‐3 day old pups. Drugs were applied to cells in serum free conditions over a range of timepoints and doses. Astrocyte conditioned media was collected from cultures and ApoE and other proteins of interest were measured by western blot.ResultWe find that inhibiting HMG‐CoA reductase, a rate‐limiting enzyme in the cholesterol synthesis pathway, with simvastatin reduces astrocyte ApoE levels in conditioned media in a dose dependent manner, while secretion of other proteins remain preserved. Inhibition of downstream cholesterol synthesis through blockade of the enzyme FDFT1 with zaragozic acid, however, has no effect on total ApoE secretion. Mice with genetic disruption of brain cholesterol metabolism also have similar levels of ApoE in CSF samples compared to control. These data suggest the effect of HMGCR inhibition on ApoE is not mediated through cholesterol. It is known that HMGCR activity is also required for maintenance of cellular protein prenylation. We tested the prenylation inhibitor FGTI‐2734 and found that it is sufficient to block astrocyte ApoE secretion, suggesting that HMGCR inhibitors may exert their effect on ApoE through this mechanism. Recent work suggests that inflammatory astrocytes exert toxic effects via neurotoxic lipids carried by ApoE and another carrier protein, CLU. We find that inhibiting HMGCR in astrocytes does not prevent cytokine‐induced conversion to an inflammatory phenotype, but remains able to block ApoE releaseConclusionWe conclude that targeted inhibition of prenylation enzymes may provide a therapeutic strategy to modulate ApoE levels in the brain.

An association of CSF apolipoprotein E glycosylation and amyloid-beta 42 in individuals who carry the APOE4 allele

Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer’s disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals (n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aβ42 levels (r = 0.53, p < 0.0001). These correlations were not observed for plasma apoE glycosylation. CSF total and secondary apoE glycosylation percentages also correlated with the concentration of CSF small high-density lipoprotein particles (s-HDL-P), which we have previously shown to be correlated with CSF Aβ42 levels and measures of cognitive function. Desialylation of apoE purified from CSF showed reduced Aβ42 degradation in microglia with E4 > E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aβ metabolism and can be a potential target of treatment.

Plasma ApoE4 Levels Are Lower than ApoE2 and ApoE3 Levels, and Not Associated with Plasma Aβ 40/42 Ratio as a Biomarker of Amyloid-β Amyloidosis in Alzheimer's Disease.

APOE4 is the strongest risk factor for Alzheimer's disease (AD). However, limited information is currently available on APOE4 and the pathological role of plasma apolipoprotein E (ApoE) 4 remains unclear. The aims of the present study were to measure plasma levels of total ApoE (tE), ApoE2, ApoE3, and ApoE4 using mass spectrometry and elucidate the relationships between plasma ApoE and blood test items. We herein examined plasma levels of tE, ApoE2, ApoE3, and ApoE4 in 498 subjects using liquid chromatograph-mass spectrometry (LC-MS/MS). Among 498 subjects, mean age was 60 years and 309 were female. tE levels were distributed as ApoE2/E3 = ApoE2/E4 >ApoE3/E3 = ApoE3/E4 >ApoE4/E4. In the heterozygous group, ApoE isoform levels were distributed as ApoE2 >ApoE3 >ApoE4. ApoE levels were not associated with aging, the plasma amyloid-β (Aβ) 40/42 ratio, or the clinical diagnosis of AD. Total cholesterol levels correlated with the level of each ApoE isoform. ApoE2 levels were associated with renal function, ApoE3 levels with low-density lipoprotein cholesterol and liver function, and ApoE4 levels with triglycerides, high-density lipoprotein cholesterol, body weight, erythropoiesis, and insulin metabolism. The present results suggest the potential of LC-MS/MS for the phenotyping and quantitation of plasma ApoE. Plasma ApoE levels are regulated in the order of ApoE2 >ApoE3 >ApoE4 and are associated with lipids and multiple metabolic pathways, but not directly with aging or AD biomarkers. The present results provide insights into the multiple pathways by which peripheral ApoE4 influences the progression of AD and atherosclerosis.

Apolipoprotein E O-glycosylation is associated with amyloid plaques and APOE genotype

Although the APOE ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (AD), the relationship between apolipoprotein (apoE) and AD pathophysiology is not yet fully understood. Relatively little is known about the apoE protein species, including post-translational modifications, that exist in the human periphery and CNS. To better understand these apoE species, we developed a LC-MS/MS assay that simultaneously quantifies both unmodified and O-glycosylated apoE peptides. The study cohort included 47 older individuals (age 75.6 ± 5.7 years [mean ± standard deviation]), including 23 individuals (49%) with cognitive impairment. Paired plasma and cerebrospinal fluid samples underwent analysis. We quantified O-glycosylation of two apoE protein residues – one in the hinge region and one in the C-terminal region – and found that glycosylation occupancy of the hinge region in the plasma was significantly correlated with plasma total apoE levels, APOE genotype and amyloid status as determined by CSF Aβ42/Aβ40. A model with plasma glycosylation occupancy, plasma total apoE concentration, and APOE genotype distinguished amyloid status with an AUROC of 0.89. These results suggest that plasma apoE glycosylation levels could be a marker of brain amyloidosis, and that apoE glycosylation may play a role in the pathophysiology of AD.

Dietary Monounsaturated and Polyunsaturated Fatty Acids are Comparable in their Effects on Hepatic Apolipoprotein mRNA Abundance and Liver Lipid Concentrations when Substituted for Saturated Fatty Acids in Cynomolgus Monkeys

Although studies have shown that saturated and polyunsaturated fats can mediate plasma lipid and apolipoprotein (apo) concentrations at the mRNA level, there is little data on the role of monounsaturated fats. We determined hepatic lipid and apo mRNA levels in 10 cynomolgus monkeys fed three diets that provided 30% of energy as fat with 0.1% cholesterol by weight and differed solely by the substitution of saturated, mono- and polyunsaturated fats as 60% of total fat energy. Total, LDL, and HDL cholesterol, as well as LDL apo B, HDL apo A-I and HDL total apo C concentrations, were reduced with the mono- and polyunsaturated fat diets relative to the saturated fat diet. Although fat saturation did not significantly affect hepatic apo A-I, B, C-II, or E mRNA abundance, hepatic apo C-III mRNA concentrations were uniformly lower (-23%, P < 0.01) with the mono- and polyunsaturated fat diets than with the saturated fat diet. Interestingly, liver triglycerides were significantly elevated with the monounsaturated fat diet relative to the saturated fat diet, but no other differences in hepatic lipids were noted among diets. Hepatic triglyceride composition was shown to reflect dietary fatty acid composition, with liver triglycerides enriched in myristic and palmitic fatty acids during the saturated fat diet, oleic acid during the monounsaturated fat diet and linoleic acid during the polyunsaturated fat diet. We conclude that dietary monounsaturated fats are comparable to polyunsaturated fats in their effects on hepatic lipid and apo mRNA levels in this species, with both unsaturated fats significantly reducing only hepatic apo C-III mRNA abundance relative to saturated fat.

Electronegative LDL Is Associated with Plaque Vulnerability in Patients with Ischemic Stroke and Carotid Atherosclerosis

Owing to the high risk of recurrence, identifying indicators of carotid plaque vulnerability in atherothrombotic ischemic stroke is essential. In this study, we aimed to identify modified LDLs and antioxidant enzymes associated with plaque vulnerability in plasma from patients with a recent ischemic stroke and carotid atherosclerosis. Patients underwent an ultrasound, a CT-angiography, and an 18F-FDG PET. A blood sample was obtained from patients (n = 64, 57.8% with stenosis ≥50%) and healthy controls (n = 24). Compared to the controls, patients showed lower levels of total cholesterol, LDL cholesterol, HDL cholesterol, apolipoprotein B (apoB), apoA-I, apoA-II, and apoE, and higher levels of apoJ. Patients showed lower platelet-activating factor acetylhydrolase (PAF-AH) and paraoxonase-1 (PON-1) enzymatic activities in HDL, and higher plasma levels of oxidized LDL (oxLDL) and electronegative LDL (LDL(-)). The only difference between patients with stenosis ≥50% and <50% was the proportion of LDL(-). In a multivariable logistic regression analysis, the levels of LDL(-), but not of oxLDL, were independently associated with the degree of carotid stenosis (OR: 5.40, CI: 1.15-25.44, p < 0.033), the presence of hypoechoic plaque (OR: 7.52, CI: 1.26-44.83, p < 0.027), and of diffuse neovessels (OR: 10.77, CI: 1.21-95.93, p < 0.033), indicating that an increased proportion of LDL(-) is associated with vulnerable atherosclerotic plaque.

Absence of Apolipoprotein E Exacerbates Prion Pathology and Promote Microglial Mediated Neurodegeneration

AbstractBackgroundBoth Alzheimer’s disease and prionoses are neurogenerative disorders, which involve misfolded protein deposition and transition of homeostatic microglia to their neurodegenerative phenotype. In prionoses, accumulation of toxic PrPSc protein constitutes the culprit of pathogenesis. Neurodegenerative phenotype microglia (MGnD) appear early in the course of disease and play opposing roles in PrPSc mediated neurodegeneration. Whilst clearance of PrPSc has a disease‐limiting effect, MGnD‐driven neuroinflammation is deleterious to neurons. Apolipoprotein (apo) E is a pleiotropic protein, which controls MGnD phagocytic and inflammatory characteristics. Despite prominent role of microglia in prionoses, involvement of apoE in their pathogenesis has not been established.MethodWild type (WT) and Apoe‐/‐ mice, both on the B6 background, were inoculated with 22L mouse adapted scrapie strain or normal brain homogenate. Neuropathological and biochemical analyses were performed 15 and 23 weeks post inoculation (WPI) on asymptomatic and overly symptomatic animals, respectively. NanoStringTM analysis of microglia and astrocytic transcriptomic markers was performed at 23WPI.ResultPrion infection of WT mice upregulates total brain apoE level and effects cell‐type shift in the apoE expression, downregulating astrocytic apoE while increasing microglial apoE 24‐folds. 22L Apoe‐/‐ mice have significantly shorter disease incubation period, higher load of spongiform changes, and increased total brain PrP and PrPSc levels compared to 22LWT mice. In 22L Apoe ‐/‐ mice astro and micro gliosis appears earlier and is more robust compared to 22LWT mice. Transcriptomic analysis shows significantly higher level of PAN and A1, but not A2 astrocytic markers and MGnD markers in 22L Apoe ‐/‐ mice compared to 22LWT animals. Microglia in 22L Apoe ‐/‐ mice feature impaired neuronal phagocytosis and produce increased level of pro‐inflammatory interleukins and cytokines. 22L Apoe ‐/‐ mice show higher neuronal loss and greater tempo of neuronal degeneration than 22LWT mice.ConclusionOur results indicate the net effect of apoE in prion pathogenesis is protective. ApoE facilitates clearance of PrPSc and dying neurons by microglia, which becomes ineffective in the absence of apoE. Remnants of disintegrating neurons promote MGnD phenotype transition and inflammatory cascade propagating the vicious cycle of neuronal death and neuroinflammation. Our data also indicate homeostatic microglia transit to the MGnD even if they do not express apoE.

Lower CSF ApoE glycosylation associates with measures of Alzheimer's disease biomarkers

AbstractBackgroundThe mechanisms of how APOE4 allele (APOE4) increases the risk of Alzheimer’s disease (AD) pathology have not been fully elucidated. In cerebrospinal fluid (CSF), apoE is heavily glycosylated but its contribution to AD pathology is not known. The objective of this study was to determine the impact of APOE genotype and cognitive status on the relative abundance of apoE protein isoforms and their specific glycosylation patterns in CSF and plasma.MethodTotal glycosylation and ApoE isoform‐specific glycosylation were analyzed in plasma and CSF from a group of older individuals (n = 106) from the USC ADRC cohort, grouped into cognitively normal, with mild cognitive impairment, and with AD dementia. We used a new mass spectrometric immunoassay that simultaneously detects the apoE isoforms and glycoforms (O‐linked GalNAc(‐Sia)‐Gal‐Sia, and various combinations thereof).ResultIn heterozygous individuals, the apoE3/E2, E4/E2, and E4/E3 isoform ratios were all significantly lower in plasma compared to CSF. For all individuals, a single O‐linked glycan was observed in plasma, while two glycans (of the same type) per apoE were observed in CSF. The ratio of glycosylated to total apoE was greater in CSF compared to plasma for all apoE isoforms. In CSF, ApoE4 was 35% and 25% less glycosylated (P&lt;0.001) than ApoE2 and ApoE3 carriers respectively. The % of secondary glycosylation was positively correlated with CSF Aβ42 levels (R=0.55, p&lt;0.001) and negatively correlated with CSF total Tau (R=‐0.38, p=0.001). Patients with AD dementia had lower % glycosylation than individuals with MCI (p=0.008).ConclusionCSF glycosylation is lower in CSF apoE4 isoform and in those with dementia, and correlate with markers of AD pathology. Ongoing experiments are delineating the effects of ApoE glycosylation on its functions. ApoE4 glycosylation may represent a newer target of treatment in AD.

Apolipoprotein E imbalance in the cerebrospinal fluid of Alzheimer’s disease patients

ObjectiveThe purpose of this study was to examine the levels of cerebrospinal fluid (CSF) apolipoprotein E (apoE) species in Alzheimer’s disease (AD) patients.MethodsWe analyzed two CSF cohorts of AD and control individuals expressing different APOE genotypes. Moreover, CSF samples from the TgF344-AD rat model were included. Samples were run in native- and SDS-PAGE under reducing or non-reducing conditions (with or without β-mercaptoethanol). Immunoprecipitation combined with mass spectrometry or western blotting analyses served to assess the identity of apoE complexes.ResultsIn TgF344-AD rats expressing a unique apoE variant resembling human apoE4, a ~35-kDa apoE monomer was identified, increasing at 16.5 months compared with wild-types. In humans, apoE isoforms form disulfide-linked dimers in CSF, except apoE4, which lacks a cysteine residue. Thus, controls showed a decrease in the apoE dimer/monomer quotient in the APOE ε3/ε4 group compared with ε3/ε3 by native electrophoresis. A major contribution of dimers was found in APOE ε3/ε4 AD cases, and, unexpectedly, dimers were also found in ε4/ε4 AD cases. Under reducing conditions, two apoE monomeric glycoforms at 36 kDa and at 34 kDa were found in all human samples. In AD patients, the amount of the 34-kDa species increased, while the 36-kDa/34-kDa quotient was lower compared with controls. Interestingly, under reducing conditions, a ~100-kDa apoE complex, the identity of which was confirmed by mass spectrometry, also appeared in human AD individuals across all APOE genotypes, suggesting the occurrence of aberrantly resistant apoE aggregates. A second independent cohort of CSF samples validated these results.ConclusionThese results indicate that despite the increase in total apoE content the apoE protein is altered in AD CSF, suggesting that function may be compromised.

The Effect of Thymoquinone on Oxidative Stress Parameters and Apolipoprotein E in Alzheimer Model in Rats

Introduction: In this study, it was aimed to investigate the effect of thymoquinone (TQ) on oxidative stress and apolipoprotein E (ApoE) in an experimental Alzheimer’s model created with AlCl₃ and D-galactose in rats. Methods: Thirty-six Wistar Albino male rats saline group (Group 1), aluminum chloride (AlCl₃) + D-galactose (D-Gal) group (Group 2), AlCl₃ + D-Gal + TQ group (Group 3) were divided into 3 groups. The study was completed with 33 rats. Group 1 was given saline intraperitoneally (i.p) for 28 days. 2nd group; D-Gal at a dose of 60 mg/kg/day and AlCl₃ at a dose of 40 mg/kg/day were given i.p. daily for 28 days. 3rd group; D-Gal at a dose of 60 mg/kg/day and AlCl₃ at a dose of 40 mg/kg/day were given i.p. daily for 28 days. Group 3 rats were given 20 mg/kg/day TQ in corn oil by gavage for 14 days. Malonyl dialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (TAS), total oxidant capacity (TOS), glutathione peroxidase (GsH-Px), and ApoE levels were determined in the blood and brain tissues of rats in all three groups. One-way ANOVA test was used in the statistical analysis of the data. Results: Means of TAS, TOS, GSH-Px, SOD, MDA, and ApoE in blood and brain tissue of all three groups (excluding ApoE in brain tissue) were different from each other and this difference was statistically significant (p < 0.05). Conclusion: In this study, TQ, it was determined that all oxidative stress parameters examined had positively affected and decreased blood tissue ApoE levels. TQ can be used as an antioxidant and curative in Alzheimer’s disease.

Plasma apolipoprotein E levels in longitudinally followed patients with mild cognitive impairment and Alzheimer’s disease

BackgroundLow levels of plasma apolipoprotein E (apoE) and presence of the APOE ε4 allele are associated with an increased risk of Alzheimer’s disease (AD). Although the increased risk of AD in APOE ε4-carriers is well-established, the protein levels have received limited attention.MethodsWe here report the total plasma apoE and apoE isoform levels at baseline from a longitudinally (24 months) followed cohort including controls (n = 39), patients with stable amnestic mild cognitive impairment during 24 months follow up (MCI-MCI, n = 30), patients with amnestic MCI (aMCI) that during follow-up were clinically diagnosed with AD with dementia (ADD) (MCI-ADD, n = 28), and patients with AD with dementia (ADD) at baseline (ADD, n = 28). We furthermore assessed associations between plasma apoE levels with cerebrospinal fluid (CSF) AD biomarkers and α-synuclein, as well as both CSF and plasma neurofilament light chain (NfL), YKL-40 and kallikrein 6.ResultsIrrespective of clinical diagnosis, the highest versus the lowest apoE levels were found in APOE ε2/ε3 versus APOE ε4/ε4 subjects, with the most prominent differences exhibited in females. Total plasma apoE levels were 32% and 21% higher in the controls versus MCI-ADD and ADD patients, respectively. Interestingly, MCI-ADD patients exhibited a 30% reduction in plasma apoE compared to MCI-MCI patients. This decrease appeared to be associated with brain amyloid-β (Aβ42) pathology regardless of disease status as assessed using the Amyloid, Tau, and Neurodegeneration (A/T/N) classification. In addition to the association between low plasma apoE and low levels of CSF Aβ42, lower apoE levels were also related to higher levels of CSF total tau (t-tau) and tau phosphorylated at Threonine 181 residue (p-tau) and NfL as well as a worse performance on the mini-mental-state-examination. In MCI-ADD patients, low levels of plasma apoE were associated with higher levels of CSF α-synuclein and kallikrein 6. No significant correlations between plasma apoE and the astrocytic inflammatory marker YKL40 were observed.ConclusionsOur results demonstrate important associations between low plasma apoE levels, Aβ pathology, and progression from aMCI to a clinical ADD diagnosis.

Effects of toxic apolipoprotein E fragments on Tau phosphorylation and cognitive impairment in neonatal mice under sevoflurane anesthesia.

BackgroundAnesthesia induces Tau phosphorylation and cognitive impairment in young, but not adult, mice. Apolipoprotein E (ApoE) may play a protective role in neuronal activity and injury repair, whereas its toxic fragments are reported to induce neurodegeneration and neurocognitive impairment in patients with Alzheimer's disease (AD). Therefore, we set out to test the hypothesis that the difference in ApoE fragments, but not the full‐length ApoE, contributes to the difference in Tau phosphorylation and neurocognitive functions following sevoflurane anesthesia in young mice.MethodsSevoflurane was administered to wild‐type (WT), ApoE‐knockout (ApoE‐KO), ApoE3‐targeted replacement (ApoE3 expresses both full‐length and fragmented ApoE), and ApoE2‐targeted replacement (ApoE2 only expresses full‐length ApoE) mice. The mRNA and protein levels of ApoE, phosphorylated Tau (pTau), and cognitive function were tested in the mice.ResultsSevoflurane anesthesia enhanced ApoE mRNA, total ApoE, full‐length ApoE, ApoE fragments, Tau phosphorylation (AT8 and PHF1), and cognitive impairment in young mice, but not in adult mice. ApoE2, but not ApoE3 or ApoE‐KO, mice showed reduced sevoflurane‐induced pTau elevation and cognitive impairment.ConclusionThese data suggest that elevated ApoE fragments rather than full‐length ApoE might be one of the underlying mechanisms of age‐dependent Tau phosphorylation and cognitive impairment in young mice following sevoflurane anesthesia.

†Efficacy and Safety of Bempedoic Acid in Patients with Metabolic Syndrome

<h3>Lead Author's Financial Disclosures</h3> MDS has received funding/grant support from Amgen, the National Institutes of Health, and Novartis, and honorarium for consultancy from Amgen, Novartis, Novo Nordisk and Regeneron. <h3>Study Funding</h3> Esperion Therapeutics, Inc. <h3>Background/Synopsis</h3> Metabolic syndrome (MetS) is associated with increased risk of cardiovascular events. Bempedoic acid (BA) is an ATP citrate lyase inhibitor that lowers LDL-C in patients with hypercholesterolemia. <h3>Objective/Purpose</h3> To assess efficacy and safety of BA in patients with and without metabolic syndrome. <h3>Methods</h3> We investigated the lipid-lowering efficacy, safety, and effect of BA on glycemic parameters and high-sensitivity C-reactive protein (hsCRP) according to baseline metabolic status using pooled data (n=3623) from 4 phase-3, randomized, double-blind studies in which patients were randomized 2:1 to BA or placebo (PBO) for 12-52 weeks. Patients with diabetes mellitus (n=1114) were excluded from the analyses. The remaining patients were categorized as having MetS (n=936; defined using modified IAS guidelines with BMI as a proxy for waist circumference) or nonMetS (n=1573). Assessments included absolute or % change from baseline to week 12 in LDL-C, other lipid parameters, hemoglobin A1c (HbA1c), fasting plasma glucose (FPG), and hsCRP; and treatment-emergent adverse events (TEAEs). <h3>Results</h3> Background statin intensity was similar between groups (Table). BA significantly lowered LDL C at week 12 vs PBO, to a greater extent in patients with MetS than nonMetS (PBO corrected % change from baseline of −22.3 MetS; −18.4 nonMetS; interaction P=.0472). BA also significantly lowered total cholesterol, non-HDL-C, and apo B % change at week 12 vs PBO to a similar extent in both subgroups. In patients with MetS, absolute reductions in HbA1c and FPG were significantly greater with BA vs PBO; changes in HbA1C and FPG were not significantly different for BA vs PBO in the nonMetS group (interaction P=.0003 and .0017, respectively). hsCRP % change from baseline was substantially lowered with BA vs PBO in both groups (insignificant interaction based on log of hsCRP absolute change). The incidence of TEAEs was comparable in both subgroups.subgroups and greater lowering of LDL-C, HbA1c, and FPG levels in patients with MetS vs nonMetS. <h3>Conclusions</h3> Overall, BA demonstrated comparable safety in both metabolic subgroups and greater lowering of LDL-C, HbA1c, and FPG levels in patients with MetS vs nonMetS.