Rosuvastatin displays anti-atherothrombotic and anti-inflammatory properties in apoE-deficient mice.

Inflammation contributes importantly to all stages of atherosclerosis, including the onset of acute thrombotic complications. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. Moreover, statins have been shown to possess several pleiotropic properties independent of cholesterol lowering in experimental settings. Based on these premises, we investigated the anti-inflammatory and anti-atherothrombotic properties of rosuvastatin in vivo, testing its effect on cholesterol and monocyte accumulation, and on adhesion molecules and tissue factor (TF) expression. ApoE-deficient female mice were fed a cholesterol-rich diet containing rosuvastatin (0, 1, 2 or 10 mg kg(-1)d(-1)) for 12 weeks. Treatment with rosuvastatin did not significantly affect either body weight gain or plasma total cholesterol (C) and triglyceride levels. However, rosuvastatin treatment dose-dependently reduced ICAM-1 expression in the aortic valves (V) (up to 40% inhibition, p<0.05) and in the proximal segment of the ascending aorta (AA) (-50%, p<0.001). Similarly, rosuvastatin inhibited VCAM-1 expression in the V (-40%) and in the AA (-35%, p<0.05). Moreover, there was a reduced accumulation of macrophages in the V in a dose-dependent and statistically significant manner (-45%, p<0.01). These anti-inflammatory effects were reflected in a reduction of cholesterol deposition in the entire aorta, both in the free and in the esterified form. Finally, the expression of tissue factor, the most potent pro-thrombogenic agent, was consistently reduced in AA by rosuvastatin treatment (-71%, p<0.001). Altogether, these data demonstrate that rosuvastatin has anti-inflammatory and anti-atherothrombotic activities in apoE-deficient mice that could translate in a beneficial effect on atherogenesis.

A structure-activity study for the inhibition of metalloproteinase-9 activity and gene expression by analogues of gallocatechin-3-gallate.

Catechins are able to modulate the gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) by reducing its release from macrophages. Gallocatechins decrease MMP-9 secretion by lowering MMP-9 promoter activity and mRNA levels. The effect appears to be dependent on some structural and stereochemical requirements. In this study, the relationship between chemical structure and activity was studied by testing the effect of analogues of (+/-)-gallocatechin-3-gallate (+/-)-GCG, selectively deprived of hydroxyl groups, on MMP-9 activity, transcription, and secretion. Our results indicate that (+/-)-GCG and (+/-)-catechin-3-gallate are characterized by a substitution pattern compatible with direct inhibition of MMP-9 activity. Conversely, when transcription was the target, (+/-)-trans-3-flavanol-3-benzoate, lacking all the hydroxyl groups, was the most effective both in lowering MMP-9 promoter activity and consequently protein secretion, and in inhibiting nuclear-factor-kappaB-driven transcription. Our results suggest that the structural requirements for enzyme inhibition are different from those necessary for targeting gene expression.

Inhibition of metalloproteinase-9 activity and gene expression by polyphenolic compounds isolated from the bark of Tristaniopsis calobuxus (Myrtaceae).

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions, and thus inhibition of MMP activity might have therapeutic potential. The methanolic extract and the identified compounds from the bark of Tristaniopsis calobuxus Brongniart & Gris (Myrtaceae) were tested on the activity, production, and gene expression of MMP-9. The extract produced a concentration-dependent inhibition (50-95% at 10-50 microg/ml) of MMP-9 activity. The inhibitory activity was retained in the ethyl acetate-soluble fraction (50-95% inhibition at 10-50 microg/ml) which also reduced the release of MMP-9 by mouse peritoneal macrophages up to 80%. In the ethyl acetate-soluble fraction, two active fractions, 5A and 5B were identified. HPLC-MS and NMR analyses of these fractions indicated the presence of gallocatechin, ellagic acid, and its glycoside derivatives. Since the absolute configuration of gallocatechin was not determined, in the next experiments both (+)-gallocatechin (2R,3S) and (-)-gallocatechin (2S,3R) were tested, and (-)-epigallocatechin (2R,3R) was included for comparison. 5A and 5B inhibited MMP-9 secretion, an observation which correlated with the decrease of MMP-9 promoter activity and the downregulation of mRNA levels. All compounds decreased MMP-9 mRNA levels and secretion. Ellagic acid, (+)-gallocatechin and (-)-epigallocatechin, but not (-)gallocatechin inhibited promoter-driven transcription. Thus configuration at C2 (R) of the flavanol seem to be critical for the interaction with the promoter.

5,6-Diphenylpyridazine derivatives as acyl-CoA:cholesterol acyltransferase inhibitors.

Alkyl-5,6-diphenylpyridazine derivatives combining several main features of ACAT inhibitors, such as a long alkyl side chain linked to a heterocycle and the o-diphenyl system, were synthesized and tested. Moreover, modeling studies on representative terms were performed. Some compounds displayed ACAT inhibition in the micromolar range, both on the enzyme isolated from rat liver microsomes and in cell-free homogenate of murine macrophages.

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.

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.

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.

The new calcium antagonist lercanidipine and its enantiomers affect major processes of atherogenesis in vitro: is calcium entry involved?

Atherosclerosis results from multiple factors and involves several mechanisms, including endothelial monocyte and smooth muscle cell (SMC) changes, cholesterol accumulation, plaque rupture and thromboembolism. Calcium ions play a role in the initial and chronic development of atherosclerotic lesions. Several studies in experimental animal models have demonstrated the potential direct antiatherosclerotic effects of calcium antagonists. In this study the antiatherogenic activity of lercanidipine, a new lipophilic, second-generation calcium antagonist, was investigated. Lercanidipine and its enantiomers inhibited the replication and migration of arterial myocytes in concentrations ranging from 10 to 50 microM. The antiproliferative effect of lercanidipine was dose dependent, with a potency similar to that of lacidipine and nifedipine, and was unrelated to the stereoselectivity of enantiomers to bind L-type calcium channels. Lercanidipine and its enantiomers (25 microM) decreased the serum-induced elevation of [Ca2+]i in SMC, with the (S)-enantiomer (69% inhibition) being 2.4-fold more active than the (R)-counterpart (29% inhibition). The studies performed with enantiomers of lercanidipine suggest that the observed effects are not related to the blockade of voltage-dependent Ca2+ channels and confirm, at least in vitro, the pharmacological potential of the compound to influence negatively the process of atherogenesis.

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. Fluvastatin's 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.

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.

Direct vascular effects of HMG-CoA reductase inhibitors.

Several studies have demonstrated that any beneficial effect of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) on coronary events are linked to their hypocholesterolemic properties. However, since mevalonic acid (MVA), the product of the enzyme reaction, is the precursor of numerous metabolites, inhibition of HMG-CoA reductase has the potential to result in pleiotropic effects. MVA and other intermediates of cholesterol synthesis (isoprenoids) are necessary for cell proliferation and other important cell functions, hence effects other than cholesterol reduction may help to explain the antiatherosclerotic properties of statins. Recently, we provided in vitro evidence that fluvastatin, simvastatin, lovastatin, cerivastatin, but not pravastatin, dose-dependently decrease smooth muscle cells (SMC) migration and proliferation, independently of their ability to reduce plasma cholesterol. Moreover, statins are able to reduce the in vitro cholesterol accumulation in macrophages, by blocking cholesterol esterification and endocytosis of modified lipoproteins. This in vitro inhibition was completely prevented by the addition of mevalonate and partially by all-trans farnesol and all-trans geranylgeraniol, confirming the specific role of isoprenoid metabolites--probably through a prenylated protein(s)--in regulating these cellular events. The inhibitory effect of lipophilic statins on SMC proliferation has been recently shown in different models of proliferating cells such as cultured arterial myocytes and rapidly proliferating carotid and femoral intimal lesions in rabbits. Finally, ex vivo studies recently showed that sera from fluvastatin-treated patients interfere with smooth muscle cell proliferation. These results suggest that HMG-CoA reductase inhibitors exert a direct antiatherosclerotic effect in the arterial wall, beyond their effects on plasma lipids, that could translate into a more significant prevention of cardiovascular disease.

Effect of lacidipine on cholesterol esterification: in vivo and in vitro studies.

1. Cholesterol esterification and accumulation in the arterial wall is a hallmark of atherogenesis. Several preclinical studies suggest that calcium antagonists may exert antiatherosclerotic activity by directly affecting atherogenesis in the arterial wall. We investigated the effect of the second generation dihydropyridine calcium antagonist lacidipine on cholesterol metabolism in vivo in the aortic arch of cholesterol fed rabbits, and in vitro in mouse cultured peritoneal macrophages. 2. Treatment of cholesterol-fed rabbits with 1, 3 and 10 mg kg-1 day-1 of lacidipine for two months reduced, in a dose-dependent manner, cholesterol esterification in the aortic arch: 24 +/- 6, 30 +/- 12, and 41 +/- 8% inhibition, respectively (P < 0.001 vs HC control). Concomitantly, drug treatment reduced total cholesterol content of the vessel wall. Lacidipine 3 and 10 mg kg-1 day-1 reduced cholesterolaemia (approximately 20%); no effect was observed at the lowest dose used (1 mg kg-1 day-1). These results suggest that the action of lacidipine on cholesterol esterification in the arterial wall involves, at least in part, a direct effect on cellular cholesterol metabolism. Inhibition of cholesterol esterification in the arterial wall was observed also in a reference group of animals treated with the specific ACAT inhibitor CI-976. 3. To evaluate the action of lacidipine on intracellular cholesterol metabolism we performed in vitro experiments with murine macrophages, the main cell type that accumulates cholesterol in the arterial wall. Lacidipine almost completely inhibited cholesterol esterification in cholesterol loaded macrophages in culture. The effect was observed independently of esterification stimuli and in cell free homogenates. The drug modified intracellular cholesterol distribution, doubling the free- to esterified sterol ratio, but did not influence the cellular rate of cholesteryl ester hydrolysis in the cell. All together these results indicate an inhibitory effect of lacidipine on cholesterol esterification catalyzed by the enzyme ACAT in murine macrophages. 4. We concluded that lacidipine influences cellular cholesterol metabolism. This effect may contribute to the potential antiatherosclerotic activity of this drug.