Lomitapide affects HDL composition and function.

BACKGROUND: Lomitapide reduces low-density lipoprotein-cholesterol (LDL-C) but also high-density lipoprotein-cholesterol (HDL-C) levels. The latter may reduce the clinical efficacy of lomitapide. We investigated the effect of lomitapide on HDL-C levels and on cholesterol efflux capacity (CEC) of HDL in patients with homozygous familial hypercholesterolemia (HoFH). METHODS AND RESULTS: Four HoFH patients were treated with increasing dosages of lomitapide. Lomitapide decreased LDL-C (range -34 to -89%). Total HDL-C levels decreased (range -16 to -34%) with a shift to buoyant HDL. ABCA1-mediated CEC decreased in all patients (range -39 to -99%). The changes of total, ABCG1- and SR-BI-mediated CEC were less consistent. CONCLUSION: Lomitapide decreased LDL-C and HDL-C levels. Our report raises the hypothesis that the anti-atherogenic potential of HDL seems to be unaffected as total CEC did not seem to change consistently. Combined with the reduction of atherogenic lipoproteins, the net effect of lomitapide appears to be beneficial in HoFH patients.

Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value.

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

The thienotriazolodiazepine Ro 11-1464 increases plasma apoA-I and promotes reverse cholesterol transport in human apoA-I transgenic mice.

BACKGROUND AND PURPOSE: Ro 11-1464 is a thienotriazolodiazepine previously described to selectively stimulate apolipoprotein A-I (apoA-I) production and mRNA level in human liver cells. Here, we studied its effects upon oral administration to human apoA-I transgenic (hapoA-I) mice. EXPERIMENTAL APPROACH: HapoA-I mice were treated for 5 days with increasing doses of Ro 11-1464. Macrophage reverse cholesterol transport (mph-RCT) was assessed by following [(3) H]-cholesterol mobilization from pre-labelled i.p. injected J774 macrophages to plasma, liver and faeces. Effects on plasma lipids, apoproteins, lecithin-cholesterol : acyltransferase (LCAT) and liver enzymes, as well as on faecal excretion of cholesterol and bile salts, and on liver lipids and mRNA contents were determined. KEY RESULTS: Treatment with Ro 11-1464 300 mg·kg(-1) ·day(-1) resulted in a nearly 2-fold increase in plasma apoA-I, a 2- to 3-fold increase in the level of large sized-pre-ß high-density lipoprotein and a 3-fold selective up-regulation of hepatic apoA-I mRNA, but a marked decrease in all plasma lipids and LCAT activity. Mpm-RCT was decreased in blood but markedly increased in faecal sterols (4-fold) and bile acids (1.7-fold). However, liver weight and liver enzymes in plasma were also increased, in parallel with an increase in liver cholesterol ester content (all these effect being significant). CONCLUSION AND IMPLICATIONS: In this model Ro 11-1464 causes increased hepatic expression and plasma levels of apoA-I and a suppression of LCAT, and a marked enhancement of reverse cholesterol transport, but also some symptoms of liver toxicity. The compound may therefore be a prototype for a next generation of anti-atherosclerotic medicines.

Hypocomplementemic type II membranoproliferative glomerulonephritis in a male patient with familial lecithin-cholesterol acyltransferase deficiency due to two different allelic mutations.

Patients with familial lecithin-cholesterol acyltransferase (LCAT) deficiency very often show progressive glomerulosclerosis with evolution to end-stage disease. High levels of an abnormal lipoprotein (lipoprotein X) cause glomerular capillary endothelial damage. The ultrastructural study of renal biopsy specimens shows characteristic glomerular deposits of membrane-like, cross-striated structures and vacuole structures. The gene encoding for LCAT has been mapped to chromosome 16q22.1, and several mutations of this gene cause LCAT deficiency which is inherited as an autosomal recessive trait and which is characterized by corneal opacities, normochromic normocytic anemia, and renal dysfunction. Herein we report clinical features and renal histological findings concerning a 24-year-old male patient with classical familial LCAT deficiency due to two different allelic mutations: a nonsense mutation inherited from the father and a missense mutation inherited from the mother. Moreover, the patient showed glomerular histological lesions and an immunofluorescent glomerular pattern typical of hypocomplementemic membranoproliferative type II glomerulonephritis (dense-deposit disease). The nature of electron-dense material that characterizes dense-deposit disease is still unknown, but there are suggestions that some chemical modifications might occur in the renal basement membranes. Therefore, this clinical case might induce to consider possible relations between disorders of the lipoprotein metabolism and renal dense-deposit disease.

A point mutation in ABC1 gene in a patient with severe premature coronary heart disease and mild clinical phenotype of Tangier disease.

The proband is a 50 year-old woman born from a consanguineous marriage. She has been suffering from angina pectoris since the age of 38 and underwent coronary bypass surgery for three-vessel disease at 48. The presence of low plasma levels of total cholesterol and high density lipoprotein (HDL) cholesterol (2.4 and 0.1 mmol/l) and apo AI (<15 mg/dl), associated with corneal lesions and a mild splenomegaly suggested the diagnosis of Tangier disease. However, none of the other features of Tangier disease, including hepatomegaly, anemia and peripheral neuropathy, were present. The analysis of the dinucleotide microsatellites located in chromosome 9q31 region demonstrated that the proband was homozygous for the alleles of D9S53, D9S1784 and D9S1832. The mother and son of the proband, both with low levels of HDL cholesterol, shared one of the proband's haplotypes, whereas neither of these haplotypes was present in the normolipidemic proband's sister. The sequence of ATP-binding cassette transporter 1 (ABC1-1) cDNA obtained by reverse transcription-PCR (RT-PCR) of total RNA isolated from cultured fibroblasts showed that the proband was homozygous for a C>T transition in exon 13, which caused a tryptophane for arginine substitution (R527W). This mutation was confirmed by direct sequencing of exon 13 amplified from genomic DNA. It can be easily screened, as the nucleotide change introduces a restriction site for the enzyme Afl III. R527W substitution occurs in a highly conserved region of the NH2 cytoplasmic domain of ABC1 protein. R527W co-segregates with the low HDL phenotype in the family and was not found in 200 chromosomes from normolipidemic individuals.

Cell cholesterol efflux to reconstituted high-density lipoproteins containing the apolipoprotein A-IMilano dimer.

The apolipoprotein A-IMilano (apoA-IM) is a molecular variant of apoA-I characterized by the Arg(173)-->Cys substitution, resulting in the formation of homodimers A-IM/A-IM. The introduction of the interchain disulfide bridge in the A-IM dimer limits the apolipoprotein conformational flexibility and restricts HDL particle size heterogeneity, thus possibly affecting HDL function in lipid metabolism and atherosclerosis protection. To investigate whether the structural changes in A-IM/A-IM affect apoA-I capacity for cell cholesterol uptake, we tested the ability of four reconstituted HDL (rHDL), that contained either apoA-I or A-IM/A-IM, to remove cholesterol from Fu5AH hepatoma cells and cholesterol-loaded murine primary macrophages (MPM). As the HDL particle size is known to affect the rHDL capacity for cell cholesterol uptake, the reconstitution conditions were carefully selected to produce two sets of rHDL particles of small and large size (7.8 and 12.5 nm in diameter). The small A-IM/A-IM rHDL were more efficient than the corresponding apoA-I particles as acceptors of membrane cholesterol from Fu5AH cells and MPM, and as inhibitors of cholesterol esterification in MPM. The large rHDL and the lipid-free apolipoproteins displayed instead similar capacities for cell cholesterol efflux. These results suggest that cell cholesterol efflux to rHDL particles of different size occurs through different mechanisms. Large HDL accommodate and retain the cholesterol molecules that have desorbed from the cell membrane into the extracellular fluid, in a process that is less sensitive to protein conformation. Small HDL accelerate the desorption of cholesterol from the cell membrane, in a process that is influenced by the conformation of the proteins on the surface of the acceptor particle. The enhanced efficiency of small A-IM/A-IM rHDL seems related to the peculiar structure of the protein on the rHDL surface, with a hydrophobic C-terminal domain extending out of the rHDL particle, available for anchoring the acceptor to the plasma membrane.

Inhibition of VCAM-1 expression in endothelial cells by reconstituted high density lipoproteins.

Plasma-derived high density lipoproteins (HDL) were found to inhibit cytokine-induced expression of endothelial cell adhesion molecules. Here we used apolipoprotein-specific reconstituted HDL (rHDL) made with phosphatidylcholine (PC) and three different apolipoproteins to identify the HDL components involved in this effect. rHDL containing apolipoprotein A-I (apoA-I), the disulfide-linked form of the apoA-IMilano variant, or apoA-II, were all effective in inhibiting the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 in TNF alpha- or LPS-stimulated HUVEC. The inhibition was concentration dependent in the range of 0.1-1.0 mg/ml (protein). PC liposomes slightly depressed TNF alpha-induced VCAM-1 expression (16% vs 43-50% for the various rHDL), whereas the lipid-free apolipoproteins had no effect. The protein component of HDL is involved in the inhibition of VCAM-1 expression in HUVEC through a rather unspecific mechanism, as three apolipoproteins with remarkably different primary structure display very similar activity.

Molecular characterization of native and recombinant apolipoprotein A-IMilano dimer. The introduction of an interchain disulfide bridge remarkably alters the physicochemical properties of apolipoprotein A-I.

The disulfide-linked dimer of apolipoprotein A-IMilano (A-IM/A-IM), a natural Arg173-->Cys variant of apoA-I, was purified from carriers' plasma and produced in Escherichia coli. The recombinant A-IM/A-IM is identical to native A-IM/A-IM, by mass spectrometry, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing. Lipid-free A-IM/A-IM undergoes concentration-dependent self-association similar to apoA-I, but at all concentrations apoA-I is more self-associated than A-IM/A-IM. Far-ultraviolet CD spectra of A-IM/A-IM reveal a highly alpha-helical structure predicted to be approximately 65% in the lipid-free and approximately 78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of alpha-helix occurs below pH 3.5 and above pH 10 in both apoA-I and A-IM/A-IM; A-IM/A-IM constantly shows a higher alpha-helical content than apoA-I over the entire pH range (1.7-12.8), suggesting that hydrophobic forces stabilize the interaction between the two A-IM chains. Indeed, and differently from apoA-I, the alpha-helical content of A-IM/A-IM is minimally affected by solvent ionic strength. The aromatic side chains in both lipid-free and lipid-bound A-IM/A-IM are immobilized in a more asymmetric and hydrophobic environment than in lipid-free apoA-I, the conformation of A-IM/A-IM being instead similar to that achieved by apoA-I following interaction with lipids. The present findings prove that rA-IM/A-IM is structurally identical to the native protein; the conformation of A-IM/A-IM is remarkably different from that of apoA-I, thus possibly explaining some of the peculiar functional properties of the apoA-IMilano dimer.

Activity profile of gemfibrozil on the major plasma lipoprotein parameters.

Gemfibrozil (G) is a widely used and highly effective fibric acid derivative. It acts both on lipoprotein disorders characterized by hypertriglyceridemia, as well as in hyperchlolesterolemia. The results of two separate studies are summarized, in order to elucidate the differential activity profile of the drug on the major plasma lipoprotein parameters. In a group of hypertriglyceridemic patients (Study I) the major activity of G was on the VLDL cholesterol (C) and triglyceride levels (respectively -40.4 and -35.7%), associated with a marked increase of the HDL-C levels, in particular in the HDL3 subfraction. In addition, apolipoprotein B levels were significantly reduced (-12%) in all treated patients. In Study II, treatment with G in severe hypercholesterolemia was compared to that with pravastatin, a major hydroxymethylglutaryl coenzyme A reductase inhibitor. G, in these patients, markedly reduced both total (-21.6%) and LDL-cholesterol (-24.4%) levels. The increase of HDL-C was less marked than in type IV patients but G, similarly to pravastatin, induced a significant reduction of apolipoprotein B, together with an increase (+19.2%) of apolipoprotein AII levels. These findings indicate that the activity profile of G is wider than expected and that it may include a reduction of LDL levels in severely hypercholesterolemic patients. The mechanism of this latter change is difficult to establish, although it may also be related to an improved delipidation of LDL, resulting in a more efficient interaction with peripheral high affinity receptors.

Predictability of low-density lipoprotein levels during apheretic treatment of hypercholesterolaemia.

The efficiency and efficacy of low-density lipoprotein (LDL) apheresis performed with a dextran sulphate cellulose (DSC) regenerating unit were tested in five familial hypercholesterolaemic patients. LDL apheresis was repeated four times at both bi-weekly and weekly intervals, processing one plasma volume each time. The efficiency of the procedure (i.e., the extent of lipoprotein removal) was nearly identical with both schedules. Efficacy parameters, i.e., decreases of plasma total and LDL cholesterol (TC and LDL-C) and apo B, were highly correlated (r greater than 0.96) with preapheresis levels, allowing an accurate prediction of the absolute lipid removal in the single individual. Plasma triglycerides, high-density lipoprotein cholesterol, apo A-I and apo A-II recovered rather rapidly, reaching 91-96% of the pre-apheresis values in 48 hours; the recovery of TC, LDL-C and apo B was much slower, with a relatively rapid early phase (80% recovery after about 7 days) followed by a successive slower rise. This pattern was highly reproducible in the single patient, allowing the definition of a simple mathematical model for an accurate (error less than 20%) prediction of the individual process. Based on this model one can design the treatment schedule necessary to maintain lipid levels within the desired range in the single individual. The hypolipidaemic efficacy of DSC apheresis appears, otherwise, not to be dependent upon the procedure per se, but on other individual factors, e.g. the amount of removable lipoproteins and the rate of lipid recovery; both can be predicted with sufficient accuracy.

Apheretic treatment of severe familial hypercholesterolemia: comparison of dextran sulfate cellulose and double membrane filtration methods for low density lipoprotein removal.

The two more widely available techniques for the extracorporeal removal of low density lipoproteins (LDL), dextran sulfate cellulose column and double membrane filtration, were comparatively tested in severe familial hypercholesterolemic patients, both acutely and during a continued 3-month treatment. The selective dextran sulfate procedure removed close to 60% of LDL and 16% of high density lipoproteins (HDL) upon each apheresis, vs. 42% and 32%, respectively, in the case of the semi-selective double membrane filtration. Upon long term biweekly treatments, LDL-cholesterol (LDL-C) decreased, with the selective procedure, from a pre-treatment level of 406.0 +/- 40.7 mg/dl to a value fluctuating between 295.4 +/- 33.8 mg/dl and 116.9 +/- 22.0 mg/dl (highest vs. lowest levels) whereas, in the case of double membrane filtration, LDL-C levels ranged between 334.8 +/- 39.8 mg/dl and 192.3 +/- 49.9 mg/dl. HDL-cholesterol levels were somewhat raised, to a higher extent with dextran sulfate apheresis. The LDL/HDL-cholesterol "atherogenic ratio", decreased from a pre-treatment value of 10.27 +/- 3.04 to values ranging between 3.61 and 6.82 with dextran sulfate and between 6.70 and 7.68 with double membrane plasmapheresis.

Alterations in the HDL system after rapid plasma cholesterol reduction by LDL-apheresis.

Changes in high density lipoprotein (HDL) subfraction structure and composition were analyzed during and after extracorporeal removal of apo B containing lipoproteins in seven familial hypercholesterolemic (FH) patients. After the apheretic procedure, carried out with dextran-sulfate-cellulose columns, the plasma levels of very low density lipoproteins (VLDL), low density lipoproteins (LDL), and HDL decreased by 72%, 50%, and 19%, respectively. The free cholesterol to esterified cholesterol ratio in plasma increased, with a 26% drop in the lecithin:cholesterol acyl transferase (LCAT) activity. In the ensuing 24 hours, VLDL, HDL, and LCAT activity approached the pretreatment levels. During this phase, possibly as a consequence of increased cholesterol esterification and exchange of cholesteryl esters for triglycerides between HDL and VLDL, HDL2a particles were detected in plasma. However, these metabolic changes did not result in clearcut modifications in the HDL2-HDL3 subfraction distribution. These findings clearly demonstrate that rapid changes in the plasma VLDL-LDL levels affect several processes involved in the HDL metabolism, but confirm that the HDL system, in spite of a considerable plasticity, displays a marked stability of the HDL2-HDL3 subfraction distribution.

Changes in high-density lipoprotein subfraction distribution and increased cholesteryl ester transfer after probucol.

The effects of probucol (500 mg twice daily) on high-density lipoprotein (HDL) subfractions and cholesteryl ester transfer from HDL to lower density lipoproteins were tested in a series of patients with Type II hypercholesterolemia. In this placebo-controlled crossover trial, patients received probucol or placebo for 8 weeks, then switched to the other agent for another 8 weeks. Probucol significantly lowered total, low-density lipoprotein and HDL cholesterol levels. HDL subfractions, separated by rate zonal ultracentrifugation, showed a dramatic reduction in HDL2, whereas changes in HDL3 were not significant. Both subfractions eluted at a characteristically lower volume, indicating a reduced flotation rate. These findings were confirmed by gradient gel electrophoretic separation, which showed a typical reduction or disappearance of HDL2b particles and the prevalence of particles in the HDL3a-HDL3b electrophoretic range in almost all patients. After treatment, cholesteryl ester transfer from HDL to lower density lipoproteins was significantly increased in all patients. These data suggest that probucol may accelerate HDL particle conversion, leading to improvement in reverse cholesterol transport from the periphery to the liver, through HDL and very low density lipoprotein.