F. Bernini

HMG-CoA Reductase Inhibitors Reduce MMP-9 Secretion by Macrophages

-Macrophages secrete matrix metalloproteinases (MMPs) that may weaken the fibrous cap of atherosclerotic plaque, predisposing its fissuration. The 92-kDa gelatinase B (MMP-9) has been identified in abdominal aortic aneurysms and in atherosclerotic tissues. Fluvastatin, through the inhibition of the isoprenoid pathway, inhibits major processes of atherogenesis in experimental models (smooth muscle cell migration and proliferation and cholesterol accumulation in macrophages). We studied the effect of fluvastatin on the activity of MMP-9 in mouse and human macrophages in culture. Conditioned media of cells treated for 24 hours with fluvastatin were analyzed by gelatin zymography. In mouse macrophages, fluvastatin (5 to 100 micromol/L) significantly inhibited in a dose-dependent manner MMP-9 activity from 20% to 40% versus control. The drug, at a concentration as low as 5 micromol/L, inhibited MMP-9 activity ( approximately 30%) in human monocyte-derived macrophages as well. Phorbol esters (TPA, 50 ng/mL) stimulated MMP-9 activity by 50%, and fluvastatin inhibited this enhanced activity up to 50% in both mouse and human macrophages. The above results on the secretion of MMP-9 were confirmed by Western blotting and ELISA. The inhibitory effect of fluvastatin was overcome by the simultaneous addition of exogenous mevalonate (100 micromol/L), a precursor of isoprenoids. Fluvastatins effect was fully reversible, and the drug did not cause any cellular toxicity. The statin did not block directly the in vitro activation of the secreted protease. Similar data were obtained with simvastatin. Altogether, our data indicate an inhibition of MMP-9 secretion by the drug. This effect is mediated by the inhibition of synthesis of mevalonate, a precursor of numerous derivatives essential for several cellular functions.

Increased Cholesterol Efflux Potential of Sera From ApoA-IMilano Carriers and Transgenic Mice

The ability of HDL to remove cholesterol from peripheral cells and drive it to the liver for excretion is believed to explain most of the strong inverse correlation between plasma HDL cholesterol levels and coronary heart disease. Carriers of the ApoA-IMilano (A-IM) mutant have a severe hypoalphalipoproteinemia but are not at increased risk for premature of coronary heart disease. To explain this apparent paradox, we compared the capacity of serum from A-IM and control subjects to extract cholesterol from Fu5AH cells. Because the A-IM carriers are all heterozygotes for the mutation, we also compared the cholesterol efflux capacity of serum from transgenic mice expressing A-IM or wild-type ApoA-I (A-IWT), in the absence of murine ApoA-I. In the whole series of human or mouse sera, cholesterol efflux was significantly correlated with several HDL-related parameters; after adjustment for concomitant variables, the only parameter that remained significantly correlated with cholesterol efflux was the serum ApoA-I concentration (r2=0.85 in humans and 0.84 in mice). The same was true when samples from control subjects, A-IM carriers, A-IWT or A-IM mice were analyzed separately. Cholesterol efflux to sera from the A-IM carriers was only reduced slightly compared with control sera (25.0+/-4.2% versus 30.4+/-3.3%), although there was a large reduction (-45%) in the serum ApoA-I concentration in the former. Cholesterol efflux was also lower to sera from A-IM than A-IWT mice (15.6+/-3.8% versus 30. 1+/-7.1%), but less than expected from the 70% reduction in serum ApoA-I concentration. A relative efflux potential of serum was calculated in each group as the slope of the regression line fitting cholesterol efflux to ApoA-I concentrations. Therefore, the relative efflux potential reflects the relative efficiency of ApoA-I in determining cell cholesterol efflux. The relative efflux potential of mouse and human sera was in the following order: A-IM mice>A-IM carriers>A-IWT mice=control subjects, suggesting a gene-dosage effect of the A-IM mutation on the efficiency of serum to extract cholesterol from cells. The high efficiency of A-IM-containing HDL for cell cholesterol uptake would result in an improved reverse cholesterol transport in the A-IM carriers, possibly explaining the low susceptibility to atherosclerosis development.

Increased synthesis of plasminogen activator inhibitor-1 by cultured human endothelial cells exposed to native and modified LDLs : an LDL receptor-independent phenomenon

The effects of native and acetylated low density lipoproteins (LDLs and acetyl-LDLs, respectively) on the release of plasminogen activator inhibitor type 1 (PAI-1) by cultured human umbilical vein endothelial cells (ECs) were evaluated. LDL and acetyl-LDL incubated with ECs for 16-18 hours increased the PAI-1 antigen levels in conditioned medium. At a concentration of 100 micrograms/mL, LDL and acetyl-LDL increased PAI-1 by 10.8 and 12.0 ng/mL, respectively (p < 0.05 and p < 0.01 versus control). The increases in PAI-1 antigen levels exerted by the lipoproteins paralleled the changes in PAI-1 activity. The effect of LDL and acetyl-LDL was concentration dependent and specific for PAI-1 because tissue-type plasminogen activator and expression of procoagulant activity were not affected by either lipoprotein. In addition, total protein synthesis evaluated in [35S] methionine-labeled ECs was not affected, and studies with cycloheximide showed that the effect of LDL and acetyl-LDL on PAI-1 release was due to de novo protein synthesis. Experiments using the C7 monoclonal antibody against the LDL receptor and binding-defective LDL indicated that the effect of LDL on the synthesis of PAI-1 was not dependent on the interaction of the LDLs with their specific receptors. Finally, extensive oxidation of LDL prevented and even reversed the effect of LDL on PAI-1 release by ECs. It is concluded that LDL specifically increases the synthesis of PAI-1 by ECs with mechanisms that are not receptor mediated.

Human Apolipoproteins A-I and A-II in Cell Cholesterol Efflux Studies With Transgenic Mice

The first step in reverse cholesterol transport is the movement of cholesterol out of cells onto lipoprotein acceptors in the interstitial fluid. The contribution of specific lipoprotein components to this process remains to be established. In this study, the role of human apolipoproteins (apo) A-I and A-II in the efflux of cellular cholesterol was investigated in transgenic mouse models in which the expression of murine apoA-I was abolished due to gene targeting (A-IKO). Serum from A-IKO mice and from mice expressing human apoA-I and/or human apoA-II was incubated with [3H]cholesterol-labeled Fu5AH rat hepatoma cells for 4 hours at 37 degrees C. The cholesterol efflux to the serum of A-IKO mice was markedly lower than that to the serum of mice transgenic for human apoA-I (5.0 +/- 1.5% versus 25.0 +/- 4.0%). Expression of human apoA-II alone did not modify the cholesterol efflux capacity of A-IKO mouse serum. Cholesterol efflux to serum of mice expressing human apoA-II together with human apoA-I was significantly lower than that to human apoA-I mouse serum (20.0 +/- 2.3% versus 25.0 +/- 4.0%). Regression analysis of cholesterol efflux versus the lipid/apolipoprotein concentrations of mouse serum suggested that 3 independent factors contribute to determine the cholesterol efflux potential of serum: the apolipoprotein composition of HDL, the serum concentration of HDL phospholipids, and the presence of a small fraction of particles containing apoA-I.

Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages

Oxidatively modified low density lipoproteins (Ox-LDL) may be involved in determining the formation of foam cells by inducing cellular cholesteryl ester accumulation. We studied the effect of copper oxidized LDL (Ox-LDL) on cholesterol accumulation and esterification in murine macrophages. Ox-LDL (44 micrograms/ml of lipoprotein cholesterol) increased the total cholesterol content of the cells from 29 to 69 micrograms/mg cell protein. Free cholesterol accounted for 85% of this increase. Acetyl LDL (Ac-LDL) (38 micrograms/ml of lipoprotein cholesterol), raised total cellular cholesterol content to a similar extent (76 micrograms/mg cell protein), however only 25% of the accumulated cholesterol was unesterified. When ACAT activity was determined after incubation of J774 cell with Ox- or Ac-LDL, Ox-LDL were 12 times less effective than Ac-LDL in stimulating cholesteryl ester formation. This was not due to an inhibition of ACAT by Ox-LDL since these lipoproteins failed to inhibit pre activated enzyme in cholesteryl ester-loaded macrophages. The uptake of 125I-Ox-LDL: was 175% that of 125I-Ac-LDL, while degradation was only 20%. All together these data suggest an altered intracellular processing of Ox-LDL, which may be responsible for free cholesterol accumulation.

Effects of calcium antagonists on lipids and atherosclerosis

The arterial accumulation of cholesterol and calcium is a hallmark of atherosclerosis. Calcium antagonists (CAs) lessen the severity of experimentally induced atherosclerosis in cholesterol-fed animals. The reduction of aortic cholesterol is one of the most striking findings. This effect is achieved without a reduction of plasma lipid or blood pressure, and is probably related to an interference of CAs with lipid metabolism in the arterial wall. To what extent these properties of CAs are due to their ability to block calcium channels still remains to be addressed. This report briefly discusses the available in vivo and in vitro evidence for the antiatherosclerotic properties of CAs, and outlines the possible mechanisms by which these compounds affect cellular lipid metabolism.

Effects of lacidipine on experimental models of atherosclerosis

Aim: To evaluate the effect of lacipine on the major processes of atherogenesis. Methods: Cell-culture methods were used to study the effect of lacidipine. Low-density lipoprotein (LDL) receptor expression and cholesterol esterification were evaluated in human skin fibroblasts and in mouse peritoneal macrophages, respectively. The effect of lacidipine on cellular proliferation was tested on aortic myocytes cultured from rat aorta. Results: Lacidipine did not affect LDL receptor expression, but it inhibited the ability of acetyl LDL to stimulate cholesterol esterification in macrophages by more than 95%. The drug inhibited cellular proliferation in a dose-dependent manner. This antiproliferative effect was confirmed in human femoral artery myocytes. In accord with the inhibitory effect on cellular growth, preliminary in vivo studies suggest that lacidipine may reduce neointimal formation induced by perivascular manipulation of the carotid artery in hypercholesterolemic rabbit. Conclusions: Our results indicate that lacidipine may be antiatherosclerotic through an effect on the major processes involved in atheroma formation.

Calcium Antagonists and Low Density Lipoprotein Receptors

The effect of different calcium antagonists on receptor-mediated LDL catabolism by human cells in culture was investigated. The calcium antagonists have been recently classified in six types, based on their pharmacological activities. The three types selective for the slow calcium channels (types I, II, and III), and the nonselective type IV have been investigated in respect to LDL metabolism. Calcium antagonists of type I (verapamil-related compounds) and type III (diltiazem) induce an increase of receptor-mediated uptake of human LDL. In contrast, calcium antagonists of type II (nifedipine-related compounds) and type IV (flunarizine) are inactive. Verapamil and diltiazem stimulate LDL receptor activity in normal fibroblasts, in fibroblasts obtained from a hypercholesterolemic type IIa heterozygous patient, in the human hepatoma cell line HepG2, but not in receptor-negative cells. The stimulatory effect depends on drug concentrations in the culture medium. Cycloheximide and alpha-amanitin prevent the stimulating effect of calcium antagonists on LDL uptake. The possible mechanisms of this action of calcium antagonists and the relationship between the in vitro stimulation of LDL receptor activity and the in vivo inhibition of lipid deposition in the arterial wall elicited by calcium antagonists are discussed. Calcium antagonists may exert part of their antiatherosclerotic activity by counteracting the inhibition of receptor-mediated lipid metabolism induced by calcium deposition in the cellular components of the arterial walls.

Lipoprotein changes and increased affinity of LDL for their receptors after acipimox treatment in hypertriglyceridemia

Modifications in plasma low and high density lipoprotein (LDL and HDL) subfraction distribution, as well as the regulation of cellular LDL metabolism by hypertriglyceridemic LDL were tested before and after treatment with acipimox, a nicotinic acid derivative, in 11 type IV hyperlipidemic patients. Large, less dense LDL particles were found in plasma after acipimox treatment, reflecting compositional changes, characterized by a 25.4% increase in cholesteryl ester content and by a 46.2% reduction of triglycerides in LDL. HDL subfractions were only slightly modified, with an increase of dense, cholesteryl ester-enriched and triglyceride poor HDL3 particles. The LDL (B,E) receptor activity in humans skin fibroblasts of LDL isolated before and after treatment was also evaluated. Hypertriglyceridemic LDL proved rather inefficient in regulating receptor activity with a close to 30% reduction vs. normal LDL in the capacity to inhibit receptor-mediated uptake and degradation of 125I-LDL. Such abnormality was fully corrected after acipimox. The reported findings indicate that acipimox treatment in type IV patients, in spite of a relatively modest plasma triglyceride reduction, can markedly modify LDL distribution and composition, normalizing the defective interaction of hypertriglyceridemic LDL with the LDL (B,E) receptor.

Ability of the LDL receptor from several animal species to recognize the human apo B binding domain: studies with LDL from familial defective apo B-100

To verify whether the LDL receptors from different animal species recognize the binding domain of human apo B-100 we studied the interaction of LDL from control and familial binding defective apo B-100 (FDB) with cultured cells. Human, monkey, bovine, guinea pig and rabbit LDL receptors distinguish between normal and binding defective LDL with a displacement ratio (defective/normal) of 3.3, 2.6, 3.4, 3.1 and 2.0, respectively. Guinea pig and rabbit receptors, however, showed affinities 2-3-fold lower than the human receptor. Hamster, rat and mouse cells failed to differentiate between normal and FDB LDL with a ratio of 1.2, 0.8, and 1.4; the apparent affinities were 4-8 times lower than that of the human receptor. The data from the latter species suggest that the LDL receptor recognizes an area of human apo B different from the human receptor binding domain. The ability of antibody Mb47 to inhibit the binding of human LDL to human, rabbit and guinea pig but not to mouse cells further stresses this concept. Moreover, in 17 alpha-ethinyl estradiol-treated rats the rate of disappearance from plasma of FDB and control 125I-labelled LDL was identical, thus confirming the in vitro observations. These data suggest that the binding domain of the LDL receptor is functionally conserved in man, monkey, cow, rabbit and guinea pig, but is quite distinct in rat, mouse and hamster.