Steven J.R. Meex

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.

Reversal of Hypoxia in Murine Atherosclerosis Prevents Necrotic Core Expansion by Enhancing Efferocytosis

Objective— Advanced murine and human plaques are hypoxic, but it remains unclear whether plaque hypoxia is causally related to atherogenesis. Here, we test the hypothesis that reversal of hypoxia in atherosclerotic plaques by breathing hyperoxic carbogen gas will prevent atherosclerosis. Approach and Results— Low-density lipoprotein receptor–deficient mice (LDLR-/-) were fed a Western-type diet, exposed to carbogen (95% O2, 5% CO2) or air, and the effect on plaque hypoxia, size, and phenotype was studied. First, the hypoxic marker pimonidazole was detected in murine LDLR-/- plaque macrophages from plaque initiation onwards. Second, the efficacy of breathing carbogen (90 minutes, single exposure) was studied. Compared with air, carbogen increased arterial blood pO2 5-fold in LDLR-/- mice and reduced plaque hypoxia in advanced plaques of the aortic root (−32%) and arch (−84%). Finally, the effect of repeated carbogen exposure on progression of atherosclerosis was studied in LDLR-/- mice fed a Western-type diet for an initial 4 weeks, followed by 4 weeks of diet and carbogen or air (both 90 min/d). Carbogen reduced plaque hypoxia (−40%), necrotic core size (−37%), and TUNEL+ (terminal uridine nick-end labeling positive) apoptotic cell content (−50%) and increased efferocytosis of apoptotic cells by cluster of differentiation 107b+ (CD107b, MAC3) macrophages (+36%) in advanced plaques of the aortic root. Plaque size, plasma cholesterol, hematopoiesis, and systemic inflammation were unchanged. In vitro, hypoxia hampered efferocytosis by bone marrow–derived macrophages, which was dependent on the receptor Mer tyrosine kinase. Conclusions— Carbogen restored murine plaque oxygenation and prevented necrotic core expansion by enhancing efferocytosis, likely via Mer tyrosine kinase. Thus, plaque hypoxia is causally related to necrotic core expansion.

Circulating cardiac troponin T exhibits a diurnal rhythm.

OBJECTIVES The goal of this study was to test the unverified assumption that chronically elevated cardiac troponin T (cTnT) levels fluctuate randomly around a homeostatic set point. BACKGROUND The introduction of high-sensitivity cardiac troponin (cTn) assays has improved sensitivity for acute myocardial infarction (AMI). However, many patients with a single positive cTn test result do not have AMI. Therefore, the diagnosis of AMI relies strongly on serial testing and interpretation of cTn kinetics. Essential in this regard is a profound understanding of the biological variation of cTn. METHODS Two studies were conducted to assess biological cTnT variation and to investigate the presence of a diurnal rhythm of cTnT. Study 1 comprised 23 male subjects with type 2 diabetes, with no acute cardiovascular disease. Serial venous blood samples were drawn over an 11-h period (8:30 am to 7:30 pm). In study 2, the presence of a diurnal cTnT rhythm was investigated by hourly sampling of 7 subjects from study 1 over 25 h. RESULTS In study 1, we observed a gradual decrease in cTnT concentrations during the day (24 ± 2%). This decrease was present in all participants and was most prominent in subjects with the highest baseline cTnT values (Pearsons R 0.93). Diurnal variation of cTnT, as assessed in study 2, was characterized by peak concentrations during morning hours (8:30 am, 17.1 ± 2.9 ng/l), gradually decreasing values during daytime (8:30 pm, 11.9 ± 1.6 ng/l), and rising concentrations during nighttime (8:30 am the next day, 16.9 ± 2.8 ng/l). CONCLUSIONS A diurnal cTnT rhythm substantiates the recommendation that all dynamic changes in cTnT should be interpreted in relation to the clinical presentation. Epidemiological studies and risk-stratification protocols with the use of cTnT may benefit from standardized sampling times. (Exercise and Glycemic Control in Type 2 Diabetes; NCT00945165).

A comprehensive review of upper reference limits reported for (high-)sensitivity cardiac troponin assays: the challenges that lie ahead

Abstract Cardiac troponins (cTn) are the preferred markers for the diagnosis of acute myocardial infarction (AMI). The guidelines recommend the use of the 99th percentile upper reference concentration of a healthy population as the diagnostic cut-off for AMI. However, a broad range of upper reference limits is still employed, complicating the diagnosis of AMI. This overview is meant to assist laboratory specialists to define an appropriate cut-off value for the diagnosis of AMI. Therefore, we provide an overview of the analytical performance and upper reference limits of seven (high-)sensitivity cTn assays: Roche high-sensitivity cTnT and ADVIA Centaur, Stratus CS, Dimension Vista, Vitros ECi, Access and Architect cTnI assays. It is shown that none of the reference populations completely met the guidelines, including those in package inserts. Forty percent of the studies collected less than the advised minimum of 300 subjects. Many studies (50%) did not report their inclusion criteria, while lower 99th percentile limits were observed when more stringent selection criteria were applied. Higher troponin cut-offs were found in men and elderly subjects, suggesting sex- and age-specific cut-offs would be considered. Therefore, there is still need for a large, rigorously screened reference population to more accurately establish cTn upper reference limits.

Direct comparison of clinical decision limits for cardiac troponin T and I

Objective The 99th percentile upper reference limit of high-sensitivity cardiac troponin (hs-cTn) from a healthy reference population is used for diagnosing acute myocardial infarction (AMI). Accepted current thresholds of hs-cTnT (Roche) and hs-cTnI (Abbott) are 14 and 26 ng/L, respectively. Since thresholds for hs-cTnT and hs-cTnI were derived from different reference cohorts it is unclear whether they are biologically equivalent. We directly assessed sex-specific and age-specific 99th percentile upper reference limits of hs-cTnT and hs-cTnI in a single reference cohort, to investigate whether current divergent thresholds of hs-cTnT and hs-cTnI stem from intrinsic assay differences or reflect cohort variation. Methods A healthy reference population was derived from a population-based cohort (the Maastricht Study: n=3451; age: 40–75 years). Individuals with diabetes mellitus, a history of cardiovascular disease, cardiac ischaemia on ECG, N-terminal pro-brain natriuretic peptide >125 ng/L or estimated glomerular filtration rate <60 mL/min/1.73 m2 were excluded. Non-parametric analyses were performed to assess 99th percentile upper reference limits. Results 1540 individuals were included in the healthy reference population (age 57±8 years, 52.4% women). Overall 99th percentile upper reference limits of hs-cTnT and hs-cTnI were 15 and 13 ng/L, respectively. Upper reference limits were higher in men than women (hs-cTnT: 16 vs 12 ng/L), (hs-cTnI: 20 vs 11 ng/L) and increased with age. Conclusions Direct comparison reveals numerically similar thresholds for hs-cTnT and hs-cTnI assays. This finding is in line with recently reported underdiagnosis of AMI with the current decision limit of 26 ng/L for hs-cTnI, especially among women. Downwards adjustment of the hs-cTnI threshold, differentiated for sex, would equalise clinical decision limits for both assays, and may prevent further underdiagnosis of AMI.

Up-regulation of CD36/FAT in preadipocytes in familial combined hyperlipidemia

Familial combined hyperlipidemia (FCHL) shows many features of the metabolic syndrome. The strong genetic component makes it an excellent model to study the genetic background of metabolic syndrome and insulin resistance. Adipose tissue is believed to contribute to, or even underlie, the FCHL phenotype and is an interesting target tissue for gene expression studies. However, interpretation of adipose tissue gene expression experiments is complex since expression differences cannot only arise as a direct consequence of a genetic trait, but may also reflect an adaptation to metabolic influences at the cellular level. In the present study, we measured gene expression levels in cultured primary human preadipocytes from FCHL and control subjects. Since isolated preadipocytes were allowed to replicate for weeks under standardized conditions, the contribution of previous metabolic influences is rather small whereas genetic defects are preserved and expressed in vitro. The main finding was up‐regulation of CD36/FAT in FCHL preadipocytes, confirmed in two independent groups of subjects, and a concomitant increase in CD36/FAT‐mediated fatty acid uptake. CD36/FAT overexpression has previously been shown to be associated with other insulin‐resistant states. The present data suggest that CD36/FAT overexpression in FCHL occurs very early in adipocyte differentiation and may be of genetic origin.

The ATF6-Met[67]Val Substitution Is Associated With Increased Plasma Cholesterol Levels

Objective—Activating transcription factor 6 (ATF6) is a sensor of the endoplasmic reticulum stress response and regulates expression of several key lipogenic genes. We used a 2-stage design to investigate whether ATF6 polymorphisms are associated with lipids in subjects at increased risk for cardiovascular disease (CVD). Methods and Results—In stage 1, 13 tag-SNPs were tested for association in Dutch samples ascertained for familial combined hyperlipidemia (FCHL) or increased risk for CVD (CVR). In stage 2, we further investigated the SNP with the strongest association from stage 1, a Methionine/Valine substitution at amino-acid 67, in Finnish FCHL families and in subjects with CVR from METSIM, a Finnish population-based cohort. The combined analysis of both stages reached region-wide significance (P=9×10−4), but this association was not seen in the entire METSIM cohort. Our functional analysis demonstrated that Valine at position 67 augments ATF6 protein and its targets Grp78 and Grp94 as well as increases luciferase expression through Grp78 promoter. Conclusions—A common nonsynonymous variant in ATF6 increases ATF6 protein levels and is associated with cholesterol levels in subjects at increased risk for CVD, but this association was not seen in a population-based cohort. Further replication is needed to confirm the role of this variant in lipids.

Upstream transcription factor 1 (USF1) in risk of type 2 diabetes: Association study in 2000 Dutch Caucasians

Type 2 diabetes shares substantial genetic and phenotypic overlap with familial combined hyperlipidemia. Upstream stimulatory factor 1 (USF1), a well-established susceptibility gene for familial combined hyperlipidemia, is postulated to be such a shared genetic determinant. We evaluated two established variants in familial combined hyperlipidemia (rs2073658 and rs3737787) for association with type 2 diabetes in two Dutch case-control samples (N=2011). The first case-control sample comprised 501 subjects with type 2 diabetes from the Breda cohort and 920 healthy blood bank donors of Dutch Caucasian origin. The second case-control sample included 211 subjects with type 2 diabetes, and 379 normoglycemic controls. SNP rs2073658 and SNP rs3737787 were in perfect linkage disequilibrium. In the first case-control sample, prevalence of the major allele was higher in patients than in controls (75% versus 71%, OR=1.25, p=0.018). A similar effect-size and -direction was observed in the second case-control sample (76% versus 72%, OR=1.22, p=0.16). A combined analysis strengthened the evidence for association (OR=1.23, p=0.006). Notably, the increased risk for type 2 diabetes could be ascribed to the major allele, and its high frequency translated to a substantial population attributable risk of 14.5%. In conclusion, the major allele of rs2073658 in the USF1 gene is associated with a modestly increased risk to develop type 2 diabetes in Dutch Caucasians, with considerable impact at the population level.

Effect of Antioxidant Supplementation on Exercise- Induced Cardiac Troponin Release in Cyclists: A Randomized Trial

Background Cardiac troponin is the biochemical gold standard to diagnose acute myocardial infarction. Interestingly however, elevated cardiac troponin concentrations are also frequently observed during and after endurance-type exercise. Oxidative stress associated with prolonged exercise has been proposed to contribute to cardiac troponin release. Therefore, the aim of this study was to assess the effect of 4 week astaxanthin supplementation (a potent cartenoid antioxidant) on antioxidant capacity and exercise-induced cardiac troponin release in cyclists. Methods Thirty-two well-trained male cyclists (age 25±5, weight 73±7 kg, maximum O2 uptake 60±5 mL·kg−1·min−1, Wmax 5.4±0.5 W·kg−1; mean ± SD) were repeatedly subjected to a laboratory based standardized exercise protocol before and after 4 weeks of astaxanthin (20 mg/day), or placebo supplementation in a double-blind randomized manner. Blood samples were obtained at baseline, at 60 min of cycling and immediately post-exercise (≈ 120 min). Results The pre-supplementation cycling trial induced a significant rise of median cardiac troponin T concentrations from 3.2 (IQR 3.0–4.2) to 4.7 ng/L (IQR 3.7–6.7), immediately post-exercise (p<0.001). Four weeks of astaxanthin supplementation significantly increased mean basal plasma astaxanthin concentrations from non-detectable values to 175±86 µg·kg−1. However, daily astaxanthin supplementation had no effect on exercise-induced cardiac troponin T release (p = 0.24), as measured by the incremental area under the curve. Furthermore, the elevation in basal plasma astaxanthin concentrations was not reflected in changes in antioxidant capacity markers (trolox equivalent antioxidant capacity, uric acid, and malondialdehyde). Markers of inflammation (high-sensitivity C-reactive protein) and exercise-induced skeletal muscle damage (creatine kinase) were equally unaffected by astaxanthin supplementation. Conclusion Despite substantial increases in plasma astaxanthin concentrations, astaxanthin supplementation did not improve antioxidant capacity in well-trained cyclists. Accordingly, exercise-induced cardiac troponin T concentrations were not affected by astaxanthin supplementation. Trial registration ClinicalTrials.gov NCT01241877

Immature platelet fraction measured on the Sysmex XN hemocytometer predicts thrombopoietic recovery after autologous stem cell transplantation

A period of thrombocytopenia is common after stem cell transplantation (SCT). To prevent serious bleeding complications, prophylactic platelet transfusions are administered. Previous studies have shown that a rise in immature platelets precedes recovery of platelet count. Our aim was to define a cutoff value for immature platelets predicting thrombopoietic recovery within 2 d.