Dynamin is involved in endolysosomal cholesterol delivery to the endoplasmic reticulum: role in cholesterol homeostasis.

Cholesterol is one of the most essential membrane components in mammalian cells and plays a critical role in several cellular functions. It is now established that intracellular cholesterol transport contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we examined the role of clathrin- and dynamin-dependent trafficking on the regulatory machinery involved in cholesterol homeostasis. Thus, expression levels of three major sterol-sensitive genes, that is sterol-regulatory element binding protein 2 (SREBP-2), hydroxymethylglutaryl-coenzyme A (HMGCoA) reductase and low-density lipoprotein (LDL) receptor, were monitored to study the cell response to the addition of LDL-derived cholesterol. We found that inhibition of clathrin-dependent endocytosis had no effect on the intracellular distribution of cholesterol and the regulation of sterol-sensitive genes. In contrast, inhibition of dynamin activity resulted in the lack of regulation of SREBP-2, HMGCoA reductase and LDL receptor genes. Immunolocalization studies along with the measure of free and esterified cholesterol indicated that dynamin inactivation led to the accumulation of free cholesterol (FC) within the late endosomal (LE)/lysosomal compartment resulting in insufficient delivery of regulatory cholesterol to the endoplasmic reticulum (ER) where the transcriptional control of sterol-sensitive genes occurs. Our data therefore indicate that dynamin plays a critical role in the delivery of cholesterol from the LE/lysosomal network to the ER and highlight the importance of LE trafficking in cholesterol homeostasis.

Enhanced efflux of cholesterol from ABCA1-expressing macrophages to serum from type IV hypertriglyceridemic subjects.

Since elevated plasma triglycerides (TGs) are an independent cardiovascular risk factor, we have compared the cholesterol efflux potential of sera from asymptomatic hypertriglyceridemic (HTG) type IIb, type IV or normolipidemic (NLP) individuals using two different cell systems. In both type IIb and IV HTG, the efflux of cholesterol from SR-BI-rich Fu5AH cells was similar to that obtained with NLP. The maintenance of efflux efficiency in spite of reduced HDL-cholesterol levels can be mainly attributed to the relative enrichment of HDL with phospholipid. In the J774 macrophage cell system, pretreatment with cAMP, which upregulates ABCA1, induced a markedly higher increase in efflux to type IV sera compared with type IIb or NLP. In addition, type IV sera exhibited two-fold higher pre-beta HDL relative concentration (percentage of total apo AI) compared with NLP. Moreover, positive correlations were established between ABCA1-mediated efflux and the serum pre-beta HDL levels or TG concentrations. Thus, the hyperTGemia is associated with a higher fraction of apo AI recovered as pre-beta HDL which appear to be partly responsible for enhanced efflux obtained upon the cAMP stimulation of J774 cells. In conclusion, we demonstrated for the first time that the ABCA1-expressing J774 cell system is responsive to the percent of apo AI present in human serum as pre-beta HDL. Our results suggest that high-plasma TG, accompanied by low HDL may not result in an impaired cholesterol efflux capacity.

Prognostic value of cardiac markers in ESRD: Chronic Hemodialysis and New Cardiac Markers Evaluation (CHANCE) study.

BACKGROUND: Cardiac disease is the main cause of mortality in long-term hemodialysis patients. Cardiac troponins (cTn) have been proposed to be markers of cardiac damage, but their value is still debated in hemodialysis patients. The aim of this prospective study is to assess the prognostic value of biochemical cardiac markers in long-term hemodialysis patients. METHODS: We measured serum levels of cTn I (cTnI), cTn T (cTnT), and creatine kinase-MB (CK-MB) in 258 asymptomatic patients (mean age, 60 +/- 15 years; 150 men) before the dialysis treatment. All causes of death and major adverse cardiac events (MACEs: cardiac death, myocardial infarction, or unstable angina) were recorded at 1 and 2 years of follow-up. A Cox proportional hazard regression model was used to identify factors predictive of mortality. RESULTS: On inclusion, 48 patients (18.6%) had cTnT levels greater than 0.1 ng/mL, 46 patients (17.8%) had cTnI levels greater than 0.15 ng/mL, and 18 patients (7.0%) had CK-MB levels greater than 3 ng/mL. Of 246 patients followed up at 2 years, 64 patients (26%) had died, including 29 patients (11.8%) of cardiac disease, and 49 patients (19.9%) experienced at least 1 MACE. MACEs were significantly greater for patients with elevated predialysis serum cTnT and CK-MB levels (>0.1 ng/mL and 3 ng/mL, respectively) than for patients with normal levels of these cardiac markers (31.9% versus 17.1%; P = 0.01; 38.9% versus 18.4%; P = 0.02, respectively). No differences were found for cTnI levels. In multivariate analysis, age (relative risk [RR], 1.04; P = 0.002), previous ischemic heart disease (RR, 2.5; P = 0.0001), and serum cTnT levels greater than 0.1 ng/mL (RR, 1.9; P = 0.04) were independent significant factors for MACEs. CONCLUSION: Increased predialysis serum levels of cTnT and CK-MB, but not cTnI, were predictive of a high risk for overall mortality and MACEs at 2 years in asymptomatic hemodialysis patients.

Characterization of polymorphic structure of SREBP-2 gene: role in atherosclerosis.

Sterol regulatory element binding proteins (SREBPs) are membrane-bound transcription factors that control the metabolism of cholesterol and fatty acids in mammalian cells. We postulated that polymorphisms (SNPs) in SREBP-2 gene might influence lipid parameters and the risk of coronary atherosclerosis. PCR-SSCP analysis and direct sequencing of DNA from 64 asymptomatic hypercholesterolemic men revealed seven genetic SREBP-2 SNPs. The genotype distribution of four of these SNPs (1668G>T, 1784G>C, 3474T>C and 3705C>T), and their influence on plasma lipid values and clinical parameters was studied in 655 asymptomatic men previously selected for the presence of at least one cardiovascular risk factor (hypertension, hypercholesterolemia, tobacco consumption). No significant relation was found with lipid parameters but there was a significant association between the 1784G>C polymorphism and intima-media thickness (IMT) measured in 497 subjects. Thus, a common variation in the SREBP-2 gene is related with early-stage carotid atherosclerosis in subjects with a risk of cardiovascular events without detectable change in plasma lipid levels.

Homocysteine decreases endothelin-1 expression by interfering with the AP-1 signaling pathway.

The increased cardiovascular risk associated with hyperhomocysteinemia has been linked to homocysteine-induced endothelial cell (EC) dysfunction. Endothelin-1 is a vasoactive peptide, synthesized mainly by vascular ECs. We have previously shown that homocysteine decreases endothelin-1 biosynthesis. Here we addressed the molecular mechanism of endothelin-1 regulation by homocysteine. Experiments with the transcription inhibitor actinomycin D indicated that the decrease in preproendothelin-1 mRNA content in homocysteine-treated cells did not result from transcript destabilization. Transient transfection assays demonstrated that homocysteine downregulated endothelin-1 at the transcriptional level by decreasing preproendothelin-1 promoter activity. Mutation of the activator protein-1 (AP-1) site of the promoter eliminated the repression induced by homocysteine. Western blot analysis showed that the homocysteine-induced decrease in promoter activity was not associated with reduced expression of the AP-1 components c-Fos and c-Jun. The inhibitory action of homocysteine on preproendothelin-1 mRNA expression was not prevented by cycloheximide. Electrophoretic mobility shift assays demonstrated that homocysteine reduced the binding activity of ECs nuclear extracts to an AP-1 consensus site. These results indicate that homocysteine downregulates endothelin-1 synthesis by inhibiting AP-1 activity, and that the AP-1 signaling pathway may be of major importance in homocysteine-induced endothelial dysfunction.

Homocysteine is the only plasma thiol associated with carotid artery remodeling.

Several authors have reported that moderate hyperhomocysteinemia is related to asymptomatic carotid arterial wall remodeling, but few data are available on other thiol compounds with potential vascular toxicity. We, therefore, investigated the relationships between major plasma thiol compounds (homocysteine, cysteine and glutathione) and the structural phenotype of the common carotid artery in a cohort of 123 subjects with no evidence of cardiovascular disease. Fasting levels of thiol compounds were measured by high-performance liquid chromatography, and arterial geometry was evaluated using high-resolution echotracking devices. In univariate regression analysis, plasma homocysteine and plasma cysteine concentrations were positively associated with carotid artery internal diameter (P=0.0001 and 0.002, respectively) and intima media thickness (P=0.003 and 0.004), but the plasma glutathione concentration was not. In multivariate analysis, plasma homocysteine was independently and positively associated with carotid artery internal diameter (P<0.005) and intima media thickness (P<0.05), but plasma cysteine was not. These data suggest that homocysteine is the only plasma thiol compound that may be considered as a risk factor for preclinical cardiovascular disease.

Opposite effects of plasma from human apolipoprotein A-II transgenic mice on cholesterol efflux from J774 macrophages and Fu5AH hepatoma cells.

Overexpression of human apolipoprotein A-II (hapo A-II) in transgenic mice (hAIItg mice) induced marked hypertriglyceridemia and low levels of plasma high density lipoprotein (HDL) with a high hapo A-II content. We sought to determine whether cholesterol efflux to plasma and HDL from these mice would be affected. In the Fu5AH cell system, plasma from hAIItg mice induced a markedly lower cholesterol efflux than did control plasma, in accordance with the dependence of efflux on HDL concentration. Moreover, HDLs from hAIItg mice were less effective acceptors than were control HDLs. In the J774 macrophage cell system, pretreatment with cAMP, which upregulates ATP binding cassette transporter 1, induced a marked increase in the efflux to hAIItg plasma as well as to purified hapo A-I and hapo A-II, whereas it had no effect on cholesterol efflux to control plasma. A strong positive correlation was established between percent cAMP stimulation of efflux and plasma hapo A-II concentration. The cAMP stimulation of efflux to hAIItg mouse plasma may be linked to the presence of pre-beta migrating HDL containing hapo A-II. Thus, despite lower HDL and apolipoprotein A-I contents, the increased ability of plasma from hAIItg mice to extract cholesterol from macrophage-like cells may have an antiatherogenic influence.