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.

The natural compound berberine positively affects macrophage functions involved in atherogenesis.

BACKGROUND AND AIMS: We investigated the effect of berberine (BBR), an alkaloid showing antiatherogenic properties beyond the cholesterol lowering capacity, on macrophage cholesterol handling upon exposure to human serum and on macrophage responses to excess free cholesterol (FC) loading. METHODS AND RESULTS: Mouse and human macrophages were utilized as cellular models. Cholesterol content was measured by a fluorimetric assay; cholesterol efflux, cytotoxicity and membrane FC distribution were evaluated by radioisotopic assays. Monocyte chemotactic protein-1 (MCP-1) secretion was measured by ELISA; membrane ruffling and macropinocytosis were visualized by confocal microscopy. Exposure of cholesterol-enriched MPM to serum in the presence of 1 µM BBR resulted in a reduction of intracellular cholesterol content twice greater than exposure to serum alone (-52%; p < 0.01 and -21%; p < 0.05), an effect not mediated by an increase of cholesterol efflux, but rather by the inhibition of cholesterol uptake from serum. Consistently, BBR inhibited in a dose-dependent manner cholesterol accumulation in human macrophages exposed to hypercholesterolemic serum. Confocal microscope analysis revealed that BBR inhibited macropinocytosis, an independent-receptor process involved in LDL internalization. Macrophage FC-enrichment increased MCP-1 release by 1.5 folds, increased cytotoxicity by 2 fold, and induced membrane ruffling; all these responses were markedly inhibited by BBR. FC-enrichment led to an increase in plasma membrane cholesterol by 4.5 folds, an effect counteracted by BBR. CONCLUSION: We showed novel potentially atheroprotective activities of BBR in macrophages, consisting in the inhibition of serum-induced cholesterol accumulation, occurring at least in part through an impairment of macropinocytosis, and of FC-induced deleterious effects.

Flow-mediated dilation, carotid wall thickness and HDL function in subjects with hyperalphalipoproteinemia.

BACKGROUND AND AIMS: The relationships between very high plasma HDLc and subclinical atherosclerosis are still a matter of debate. METHODS AND RESULTS: Twenty subjects with primary hyperalphalipoproteinemia (HAL, with HDLc in the highest 10th percentile and absence of overt secondary causes of this condition), aged 30-65 years, were compared with 20 age and sex-matched controls. Lipid determination, lipoprotein particle distribution (Lipoprint(®)), Cholesterol Efflux Capacity (CEC), plasma adhesion molecule, analyses of CETP, SRB1 and LIPG genes and of different markers of subclinical vascular disease (ankle-brachial index, ABI; carotid intima-media thickness, cIMT; brachial-artery flow mediated dilation, FMD) were performed. Fasting HDLc levels were 40 mg/dl higher in HAL subjects while LDLc concentration was comparable to control group. CETP gene analysis in HAL subjects identified one novel rare Single Nucleotide Polymorphism (SNP, Asp131Asn), possibly damaging, while the common SNP p.Val422Ile was highly prevalent (50% vs. 27.4% in a control population). No rare mutations associated with HAL were found in SR-B1 and LIPG genes. Polyacrylamide gel electrophoresis in HAL subjects disclosed larger and more buoyant HDL particles than in controls, while LDL profile was much more similar. ABI, cIMT and arterial plaques did not differ in cases and controls and the two groups showed comparable FMD at brachial artery examination. Similarly, ABCA1 and ABCG1 HDL-mediated CEC, the most relevant for atheroprotection, did not discriminate between the groups and only ABCG1 pathway seemed somewhat related to arterial reactivity. CONCLUSIONS: HDL dimension, function and genetics seem scarcely related to subclinical atherosclerosis and vascular reactivity in middle-aged HAL subjects.

Cellular cholesterol efflux and cholesterol loading capacity of serum: effects of LDL-apheresis.

High LDL-cholesterol (LDL-C) characterizes familial hypercholesterolemia (FH) and familial combined hyperlipidemia (FCH). LDL-apheresis, used in these patients to reduce LDL-C levels, has been shown to also affect HDL levels and composition. We studied LDL-apheresis effects on six FH and nine FCH subjects' serum capacity to modulate cellular cholesterol efflux, an index of HDL functionality, and to load macrophages with cholesterol. Serum cholesterol efflux capacity (CEC) and macrophage cholesterol loading capacity (CLC) were measured before, immediately after, and two days after LDL-apheresis. The procedure reduced total cholesterol (TC), LDL-C, and apoB plasma levels (-69%, -80% and -74%, respectively), parameters only partially restored two days later. HDL-C and apoA-I plasma levels, reduced after LDL-apheresis (-27% and -16%, respectively), were restored to almost normal levels two days later. LDL-apheresis reduced serum aqueous diffusion (AD) CEC, SR-BI-CEC, and ABCA1-CEC. AD and SR-BI were fully restored whereas ABCA1-CEC remained low two days later. Sera immediately and two days after LDL-apheresis had a lower CLC than pre-LDL-apheresis sera. In conclusion, LDL-apheresis transiently reduces HDL-C levels and serum CEC, but it also reduces also serum capacity to deliver cholesterol to macrophages. Despite a potentially negative effect on HDL levels and composition, LDL-apheresis may counteract foam cells formation.

Cellular cholesterol flux studies: methodological considerations.

Reverse cholesterol transport (RCT) is the process in which peripheral cells release cholesterol to an extracellular acceptor such as high-density lipoprotein (HDL) which then mediates cholesterol delivery to the liver for excretion. RCT represents a physiological mechanism by which peripheral tissues are protected against excessive accumulation of cholesterol. The first step in RCT is the interaction of the cell with lipoprotein particles, a process that results in both the cellular uptake and release of cholesterol. The various components of this cholesterol flux can be viewed as efflux, influx and net flux. Experimental protocols for measuring each of these components of cholesterol flux are very different, and a number of considerations are required to design experimental approaches for the quantitation of flux parameters. Although many flux studies have been conducted in the past, the recent discoveries of the scavenger receptor B1 (SR-B1) and ATP binding cassette 1 (ABCA1), which mediate the movement of cholesterol between cells and extracellular acceptors, has led to increased interest in studies of cellular cholesterol flux. The aim of this review is to present a discussion of the methodological considerations that should be evaluated during the design and analysis of cellular cholesterol flux experiments.

Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL.

Results from several laboratories clearly indicate that expression of scavenger receptor class B type I (SR-BI) enhances the bidirectional flux of cholesterol between cells and lipoproteins. Because the activity of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis, is regulated by cell cholesterol content, we designed experiments to investigate the effect of SR-BI expression on the activity of this enzyme and on net cellular cholesterol mass. In addition, we compared the function of SR-BI with its human homolog, CD36 and LIMPII analogous 1. Our experiments demonstrate that both receptors enhance the flux of unesterified or free cholesterol bidirectionally, down a concentration gradient. Receptor-mediated cholesterol flux can effectively modulate multiple aspects of cellular cholesterol metabolism, including the pool that regulates the activity of HMG-CoA reductase. We also found that constitutive expression of SR-BI alters the steady state level of cellular cholesterol and phospholipid when SR-BI-expressing cells are maintained in medium containing serum lipoproteins. All of these effects are proportional to the level of receptor on the cell surface. These data indicate that the level of SR-BI expression determines both the rate of free cholesterol flux and the steady state level of cellular cholesterol.

Lacidipine [correction of Lalsoacidipine] modulates the secretion of matrix metalloproteinase-9 by human macrophages.

Activated macrophages within the arterial wall secrete matrix-degrading metalloproteinases (MMPs) that weaken the atherosclerotic plaque and contribute to its fissuration. Preclinical studies have shown that calcium antagonists may reduce atherogenesis in the arterial wall. In the present study we evaluated the effect of lacidipine on 92-kDa gelatinase B (MMP-9) expression in human macrophages in cultures. Cells were treated for 24 h with lacidipine and the conditioned media were analyzed. Lacidipine (1-20 microM) significantly reduced, in a dose-dependent manner, MMP-9 potential gelatinolytic capacity up to 50%. When MMP-9 expression was stimulated by treatment with phorbol esters or tumor necrosis factor-alpha, lacidipine was able to inhibit this enhanced gelatinolytic capacity up to 50 and 60%, respectively. Western blot analysis and enzyme-linked immunosorbent assay showed a reduction of MMP-9 protein actually released by cells. The addition of lacidipine in the incubation media determined no significant variation in Ca(2+) concentration. The drug did not affect MMP-9 mRNA levels, but it effectively reduced the amount of both active and total free MMP-9 secreted by human macrophages. Lacidipine reduced also the secretion of the tissue inhibitor of metalloproteinase-1 (TIMP-1); however we observed an overall reduction of the gelatinolytic activity of the cells. Finally, peritoneal macrophages, obtained from mice treated with lacidipine, showed a reduced secretion of MMP-9. Together, our data indicate that lacidipine may potentially exert an antiatherosclerotic activity by modulating the secretion of MMP-9 by macrophages. This, in addition to the previously demonstrated inhibition of cholesterol esterification, may contribute to increase plaque stability.

Cellular toxicity of N-substituted 2,2'-dicarboxamidodiphenyldisulphides with high antimicrobial activity.

In the present paper we evaluate the cellular toxicity of some N-substituted 2,2'-dicarboxamidodiphenyldisulphides with high antimicrobial activities, in view of their potential application in humans or animals. The toxicological studies have been conducted in the murine cell line of 3T3 fibroblasts as eucaryotic cellular model. Our results have allowed the identification of a series of derivatives exhibiting antimicrobial activity and low cellular toxicity. Structure-cytotoxic activity relationships are also discussed.