Image_1_HDL metabolism and functions impacting on cell cholesterol homeostasis are specifically altered in patients with abdominal aortic aneurysm.jpeg

BackgroundThe etiopathogenesis of abdominal aortic aneurysm (AAA) is still unclarified, but vascular inflammation and matrix metalloproteases activation have a recognized role in AAA development and progression. Circulating lipoproteins are involved in tissue inflammation and repair, particularly through the regulation of intracellular cholesterol, whose excess is associated to cell damage and proinflammatory activation. We analyzed lipoprotein metabolism and function in AAA and in control vasculopathic patients, to highlight possible non-atherosclerosis-related, specific abnormalities.MethodsWe measured fluorometrically serum esterified/total cholesterol ratio, as an index of lecithin-cholesterol acyltransferase (LCAT) activity, and cholesteryl ester transfer protein (CETP) activity in patients referred to vascular surgery either for AAA (n=30) or stenotic aortic/peripheral atherosclerosis (n=21) having similar burden of cardiovascular risk factors and disease. We measured high-density lipoprotein (HDL)-cholesterol efflux capacity (CEC), through the ATP-binding cassette G1 (ABCG1) and A1 (ABCA1) pathways and serum cell cholesterol loading capacity (CLC), by radioisotopic and fluorimetric methods, respectively.ResultsWe found higher LCAT (+23%; p ConclusionsWe demonstrated that compared to patients with stenotic atherosclerosis, patients with AAA had altered HDL metabolism and functions involved in their anti-inflammatory and tissue repair activity, particularly through the ABCG1-related intracellular signaling. Clarifying the relevance of this mechanism for AAA evolution might help in developing new diagnostic parameters and therapeutic targets for the early management of this condition.</p

Effects of HDL isolated from carriers of CETP mutations and controls on eNOS activation in HUVEC.

<p>Cells were incubated for 10-sex matches controls (n = 8), at the concentration of 1.0 mg of protein/ml. Western blot analysis of the phosphorylated and total forms of eNOS was performed, and the phosphorylated/total eNOS ratios were calculated by densitometric analysis and expressed as fold of increase in treated vs. untreated cells. Data points for each study participant are shown. cells. Results are mean±SEM of 3 separate experiments performed with 1 preparation of homozygote HDL2, 3 preparations of control HDL2, and 3 batches of cells. *<i>P</i><0.05 vs. untreated homozygote HDL2.</p

GGE analysis of purified HDL2 and HDL3.

<p>HDL fractions isolated from the homozygote, and a representative heterozygote and control were analyzed by GGE.</p

Hepatic ACAT2 Knock Down Increases ABCA1 and Modifies HDL Metabolism in Mice

<div><p>Objectives</p><p>ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism.</p><p>Design</p><p>WT and LXRα/β double knockout (DOKO) mice were fed a western-type diet for 8 weeks. Animals were i.p. injected with an antisense oligonucleotide targeted to hepatic ACAT2 (ASO6), or with an ASO control. Injections started 4 weeks after, or concomitantly with, the beginning of the diet.</p><p>Results</p><p>ASO6 reduced liver cholesteryl esters, while not inducing UC accumulation. ASO6 increased hepatic ABCA1 protein independently of the diet conditions. ASO6 affected HDL lipids (increased UC) only in DOKO, while it increased apoE-containing HDL in both genotypes. In WT mice ASO6 led to the appearance of large HDL enriched in apoAI and apoE.</p><p>Conclusions</p><p>The use of ASO6 revealed a new pathway by which the liver may contribute to HDL metabolism in mice. ACAT2 seems to be a hepatic player affecting the cholesterol fluxes fated to VLDL or to HDL, the latter via up-regulation of ABCA1.</p></div

2D electrophoresis analysis of purified HDL.

<p>HDL isolated from the homozygote, and a representative heterozygote and control were separated by 2D electrophoresis and immunodetected with anti apoA-I and anti apoE antibodies.</p

Effects of HDL isolated from carriers of CETP mutations and controls on VCAM-1 expression in TNFα-stimulated HUVEC.

<p>Cells were incubated overnight with HDL, HDL2, or HDL3 isolated from 7 heterozygous carriers of CETP mutations and age-sex matched controls (n = 8), at the concentration of 1.0 mg of protein/ml, before stimulation with TNFα for 8 hours. Results are expressed as percentage of VCAM-1 concentration in conditioned media of untreated TNFα-stimulated cells. Data points for each study participant are shown.</p

Sphingosine-1-phosphate levels in HDL, HDL2, and HDL3.

<p>Data are expressed as mean±SD.</p

Effect of apoE depletion on eNOS expression in HUVEC.

<p>Panel A. HDL2 isolated from the homozygous carrier of the R37X CETP mutation were separated by 2D electrophoresis, followed by anti apoA-I or anti apoE immunodetection, before (−) and after (+) incubation with heparin-MnCl₂. Panel B. Cells were incubated overnight with HDL2 (1 mg/ml) from the homozygous carrier of the R37X CETP mutation and from controls (n = 3) before (full bars) and after (open bars) treatment with heparin-MnCl₂. Western blot analysis of eNOS protein was performed, and eNOS protein band intensities were normalized for β-actin values and expressed as fold of increase in treated vs. untreated.</p

Lipid quantification in serum and HDL lipoproteins.

<p>Serum lipoproteins were separated by size exclusion chromatography, and the concentration of total (TC) and unesterified (UC) cholesterol, phospholipids (PL), and triglycerides (TG) was determined by a system for on-line detection. Data are expressed as average ± SEM (n = 6–8). Mann Whitney test, *p<0.05, † p<0.01, ‡ p<0.005.</p

Hepatic ACAT2 down regulation increases the ABCA1 protein independently of the mRNA expression.

<p>Liver membrane proteins were pooled group-wise, loaded, and separated on Tris-Acetate Gels. After transfer onto nitrocellulose membrane, samples were incubated with anti-mouse ABCA1 antibody. ABCA1 band (≈ 250 kD) was detected by chemiluminescence, and signals were plotted by μg-loaded protein. The slope of the curves was calculated by method of least square, and the slope of the ASOctrl group was set equal to 100%. Hepatic <i>Abca1</i> mRNA was quantified by real-time RT-PCR. Data were standardized for <i>Tfiib</i> mRNA expression, and normalized to WT Ctrl in each experiment. In the mRNA data error bars represent the median. Mann Whitney test, ** p< 0.01.</p